US11897926B2 - GIPR-agonist compounds - Google Patents

GIPR-agonist compounds Download PDF

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US11897926B2
US11897926B2 US17/573,317 US202217573317A US11897926B2 US 11897926 B2 US11897926 B2 US 11897926B2 US 202217573317 A US202217573317 A US 202217573317A US 11897926 B2 US11897926 B2 US 11897926B2
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ethoxy
aib
amino
glu
acetyl
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US20220127315A1 (en
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Jorge Alsina-Fernandez
Andrea Renee GEISER
Lili GUO
Samantha Grace Lyons KEYSER
John Lee
Hongchang Qu
William Christopher ROELL
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Eli Lilly and Co
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds having activity at the human glucose-dependent insulinotropic polypeptide (GIP) receptor.
  • GIP human glucose-dependent insulinotropic polypeptide
  • the present invention also relates to compounds having an extended duration of action at the GIP receptor.
  • Compounds may be useful in the treatment of type 2 diabetes mellitus (“T2DM”). Also, the compounds may be useful in the treatment of obesity.
  • T2DM is the most common form of diabetes, accounting for approximately 90% of all diabetes.
  • T2DM is characterized by high blood glucose levels associated mainly with insulin resistance.
  • the current standard of care for T2DM includes diet and exercise, treatment with oral medications, and injectable glucose-lowering drugs, including incretin-based therapies such as GLP-1 receptor agonists.
  • GLP-1 receptor agonists are currently available for treatment of T2DM, although currently marketed GLP-1 receptor agonists are generally dose-limited by gastrointestinal side effects such as nausea and vomiting.
  • Subcutaneous injection is the typical route of administration for the available GLP-1 receptor agonists.
  • oral medications and incretin-based therapies are insufficient, insulin treatment is considered.
  • T2DM is unable to reach their glycemic control goals.
  • Uncontrolled diabetes leads to several conditions associated with increased morbidity and mortality of patients.
  • Obesity is a complex medical disorder resulting in excessive accumulation of adipose tissue mass.
  • Today obesity is a global public health concern that is associated with undesired health outcomes and morbidities. Desired treatments for patients with obesity should reduce excess body weight, improve obesity-related co-morbidities, and maintain long-term weight reduction. Available treatments for obesity are particularly unsatisfactory for patients with severe obesity. There is a need for alternative treatment options to induce therapeutic weight loss in patients in need of such treatment.
  • WO2016/111971 describes peptides stated to have GLP-1R and GIPR agonist activities.
  • WO2013/164483 also discloses compounds stated to have GLP-1R and GIPR activities.
  • WO2018/181864 discloses compounds stated to have GIPR agonist activity.
  • T2DM treatments capable of providing effective glucose control for a larger portion of the patients in need of such treatment.
  • T2DM treatments capable of providing effective glucose control and with a favorable side effect profile.
  • alternate treatment options to provide therapeutic weight loss in a patient in need of such treatment, such as a patient with severe obesity.
  • diabetes treatment options may be combined with insulin therapy and/or incretin therapy to provide the patient with superior glycemic outcomes and/or more desirable side effect profiles.
  • Compounds with extended duration of action at the GIP receptor are desirable to allow for less frequent dosing of the compound.
  • embodiment 1 is a compound of Formula I Z 1 X 1 X 2 EGTX 6 ISDYSIX 13 LDX 16 X 17 X 18 QX 20 X 21 X 22 VX 24 X 25 X 26 L X 28 X 29 GPSSGAPPPSZ 2 (SEQ ID NO:4) wherein:
  • An embodiment 2 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein Z 1 is absent and X 1 is Y.
  • An embodiment 3 provides a compound, or pharmaceutically acceptable salt thereof, of embodiment 1 or embodiment 2 wherein X 2 is Aib.
  • An embodiment 4 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 3 wherein:
  • X 6 is selected from the group consisting of F and ⁇ MeF(2F).
  • An embodiment 5 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 4 wherein:
  • X 13 is selected from the group consisting of L and ⁇ MeL.
  • An embodiment 6 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 5 wherein X 16 is K or Orn.
  • An embodiment 7 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 6 wherein X 16 is K.
  • An embodiment 8 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 7 wherein X 18 is H.
  • An embodiment 9 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 8 wherein X 20 is Aib; and X 22 is F.
  • An embodiment 10 provides a compound, or a pharmaceutically acceptable salt thereof, as claimed of any one of embodiments 1 to 9 wherein X 21 is D.
  • An embodiment 11 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 10 wherein X 25 is 4-Pal or Y.
  • An embodiment 12 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 11 wherein:
  • An embodiment 13 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 11 wherein:
  • An embodiment 14 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 13 wherein q is 16.
  • An embodiment 15 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 13 wherein q is 18.
  • An embodiment 16 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein: Z 1 is absent;
  • An embodiment 17 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 16 wherein:
  • An embodiment 18 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 17 wherein: X 17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -( ⁇ -Glu)-CO—(CH 2 ) q —CO 2 H; and
  • X 26 is L.
  • An embodiment 19 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 17 wherein: X 17 is I; and X 26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -( ⁇ -Glu)-CO—(CH 2 ) q —CO 2 H.
  • An embodiment 20 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
  • An embodiment 21 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:5.
  • An embodiment 22 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:6.
  • An embodiment 23 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:7.
  • An embodiment 24 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:8.
  • An embodiment 25 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:9.
  • An embodiment 26 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:10.
  • An embodiment 27 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ NO:11.
  • An embodiment provides a method of treating a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome, comprising administering to a subject in need thereof, an effective amount of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof.
  • An embodiment provides a method of treating a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome, comprising administering to a subject in need thereof, an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.
  • An embodiment provides a method for providing therapeutic weight loss comprising administering to a subject in need thereof, an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • An embodiment is a method of treating T2DM comprising administering to a subject in need thereof, and effective amount of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof.
  • An embodiment provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy.
  • An embodiment provides a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, for use in therapy to treat a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome.
  • An embodiment provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy to treat a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome.
  • the condition is T2DM.
  • the condition is obesity.
  • the condition is type 1 diabetes (T1DM).
  • the condition is diabetes in a patient receiving insulin therapy.
  • the condition is metabolic syndrome.
  • the compounds of Formula I, or a pharmaceutically acceptable salt thereof may be useful in the treatment of a variety of symptoms or disorders.
  • certain embodiments provide a method for treatment of T2DM in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • a method for treatment of obesity in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • the method is inducing non-therapeutic weight loss in a subject, comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a method for treatment of metabolic syndrome in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • the method is treatment of diabetes in a patient receiving insulin treatment, comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • a compound of the present invention is provided in a fixed dose combination with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM.
  • agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM.
  • a compound of the present invention is provided in a fixed dose combination with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1-glucagon receptor agonist including OXM and analogs thereof.
  • agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin, an
  • a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM in the treatment of a condition selected from the group consisting of T2DM and obesity.
  • one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM in the treatment of a condition selected from the group consisting of T2DM and obesity.
  • a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1 glucagon receptor agonist including OXM and analogs of, in the treatment of a condition selected from the group consisting of T2DM and obesity.
  • agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SG
  • a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, and a SGLT-2 inhibitor in the treatment of a condition selected from the group consisting of T2DM and obesity.
  • an embodiment is a method for treating diabetes in a patient receiving insulin therapy, comprising administering an effective amount of a Compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient in need thereof.
  • An embodiment is treatment to a patient administered insulin therapy for T1DM.
  • An embodiment is treatment to patient administered insulin therapy for T2DM.
  • An embodiment is once weekly dosing.
  • An embodiment is subcutaneous treatment once weekly to a patient administered insulin therapy.
  • the insulin therapy comprises basal insulin therapy.
  • the insulin therapy comprises mealtime insulin therapy.
  • the insulin therapy comprises ultra-rapid insulin therapy.
  • Insulin therapy administered with acute infusions of a compound of Formula I, or a pharmaceutically acceptable salt thereof may enhance glucagon excursion in patients undergoing hypoglycemic clamp, thus enhancing the body's natural defense against hypoglycemia.
  • a compound of Formula I, or a pharmaceutically acceptable salt thereof can be dosed once weekly independent of the type of insulin used or doses of insulin used.
  • An embodiment is a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, administered as an effective amount to a patient receiving insulin therapy, independent of the type of insulin used or doses of insulin used.
  • An embodiment is a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, dosed once weekly as an effective amount to a patient receiving insulin therapy independent of the type of insulin used or doses of insulin used.
  • insulin therapy means treatment of a patient with diabetes using an approved insulin treatment. Such insulin therapy is known to the skilled artisan and/or clinical health care professional.
  • insulin therapy may comprise treatment using basal insulin.
  • basal insulin insulin therapy
  • mealtime insulin means insulin and/or modified insulin to be administered with meals, for example, but not limited to, insulin lispro.
  • basic insulin means modified insulin with a longer duration of action, such as, for example, but not limited to, insulin glargine.
  • Another embodiment provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome.
  • An embodiment provides the use of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome.
  • the medicament is for the treatment of T2DM.
  • the medicament is for the treatment of obesity.
  • the medicament is for use in the treatment of diabetes in a patient receiving insulin therapy.
  • Another embodiment provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and at least one selected from the group consisting of a carrier, diluent, and excipient.
  • a pharmaceutical composition for subcutaneous administration is provided.
  • treating includes restraining, slowing, stopping, or reversing the progression or severity of a symptom, condition, or disorder.
  • Certain compounds of the present invention are generally effective over a wide dosage range.
  • dosages for once weekly parenteral dosing may fall within the range of 0.05 mg to about 60 mg per person per week.
  • the compounds of Formula I, or a pharmaceutically acceptable salt thereof, include novel amino acid sequences having affinity for the GIP receptor, with desired potency at the receptor.
  • GIP is a 42 amino acid peptide (SEQ ID NO:1), which, like GLP-1, is known as an incretin, and it plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose.
  • GLP-1 is a 36 amino acid peptide, the major biologically active fragment of which is produced as a 30-amino acid, C-terminal amidated peptide (GLP-1 7-36 ) (SEQ ID NO:2).
  • Glucagon is a 29-amino acid peptide hormone (SEQ ID NO:3) secreted by ⁇ -cells of the islet of Langerhans in the pancreas and is involved in glucose homeostasis.
  • the compounds of present invention provide desired potency at the GIP receptor with high degree of selectivity against the GLP-1R and the Glucagon receptor.
  • compounds have desired GIP receptor activity with extended duration of action.
  • amino acid means both naturally occurring amino acids and unnatural amino acids.
  • alpha-amino isobutyric acid or “Aib,” “4Pal,” “Orn,” and the like.
  • Orn means L-ornithine.
  • 4Pal means 3-(4-Pyridyl)-L-alanine.
  • ⁇ MeF(2F) means alpha-methyl 2-fluoro-L-phenylalanine.
  • ⁇ MeY and “ ⁇ MeL” mean alpha-methyl-L-tyrosine and alpha-methyl-L-leucine, respectively.
  • e and D-Glu mean D-glutamic acid.
  • D-Tyr and “y” each mean D-tyrosine.
  • D-Ala and a each mean D-alanine.
  • ⁇ MeF means alpha-methyl-F and alpha-methyl-Phe.
  • Iva means L-isovaline.
  • the conjugation is an acylation. In an embodiment, the conjugation is to the epsilon-amino group of the K side-chain.
  • a fatty acid moiety is conjugated, via a linker, to a K at position 17. In an embodiment of the compounds of the present invention, a fatty acid moiety is conjugated, via a linker, to a K at position 26.
  • q is selected from the group consisting of 16 and 18. In an embodiment, q is 16. In an embodiment, q is 18.
  • the terms “activity,” “activate[s]” “activat[ing]” and the like refers to the capacity of a compound, or a pharmaceutically acceptable salt thereof, to bind to and induce a response at the receptor, as measured using assays known in the art, such as the in vitro assays described below.
  • the affinity of compounds of Formula I, or a pharmaceutically acceptable salt thereof, for the GIP receptor may be measured using techniques known for measuring receptor binding levels in the art, including, for example, those described in the examples below, and is commonly expressed as a K i value.
  • the activity of the compounds of the present invention at the GIP receptor may also be measured using techniques known in the art, including for example the in vitro activity assays described below, and is commonly expressed as an EC 50 value, which is the concentration of compound causing half-maximal simulation in a dose response curve.
  • a pharmaceutical composition of a compound of Formula I is suitable for administration by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal).
  • parenteral route e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal.
  • Compounds of the present invention may react with any of a number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts.
  • Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. (See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)).
  • Pharmaceutically acceptable salts of the present invention include, but are not limited to, sodium, trifluoroacetate, hydrochloride, ammonium, and acetate salts.
  • a pharmaceutically acceptable salt is selected from the group consisting of sodium, hydrochloride, and acetate salts.
  • the present invention also encompasses novel intermediates and processes useful for the synthesis of compounds of the present invention, or a pharmaceutically acceptable salt thereof.
  • the intermediates and compounds of the present invention may be prepared by a variety of procedures known in the art. In particular, the Examples below describe a process using chemical synthesis. The specific synthetic steps for each of the routes described may be combined in different ways to prepare compounds of the present invention.
  • the reagents and starting materials are readily available to one of ordinary skill in the art.
  • the term “effective amount” refers to the amount or dose of a compound of the present invention, or a pharmaceutically acceptable salt thereof, which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment.
  • An effective amount can be determined by a person of skill in the art using known techniques and by observing results obtained under analogous circumstances.
  • a number of factors are considered, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
  • the term “subject in need thereof” refers to a mammal, preferably a human, with a disease or condition requiting treatment or therapy, including for example those listed in the preceding paragraphs.
  • EDTA means ethylenediaminetetraacetic acid.
  • DMSO dimethyl sulfoxide.
  • CPM counts per minute.
  • IBMX 3-isobutyl-1-methylxanthine.
  • LC/MS means liquid chromatography/mass spectrometry.
  • HTRF means homogeneous time-resolved fluorescence.
  • DMF refers to N,N-dimethylformamide.
  • DCM refers to dichloromethane.
  • TFA refers to trifluoroacetic acid.
  • TFA salt refers to trifluoroacetate salt.
  • RP-HPLC means reversed-phase high performance liquid chromatography.
  • SEQ ID NO:5 The structure of SEQ ID NO:5 is depicted below using the standard single letter amino acid codes with the exceptions of residues D-Ala2, K17, Aib20, and Ser39, where the structures of these amino acid residues have been expanded:
  • Example 1 The peptide backbone of Example 1 is synthesized using fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl (t-Bu) chemistry on a Symphony multiplex peptide synthesizer (Gyros Protein Technologies. Arlington, AZ; 3.3.0.1), software version 3.3.0.
  • the resin consists of aminomethyl polystyrene functionalized with a Rink Amide linker (polystyrene AM RAM, RAPP polymeric GmbH, H40023, 200-400 mesh) at a substitution of 0.8 mmol/g.
  • Standard side-chain protecting groups are used with the following exceptions: Fmoc-Lys(Mtt)-OH, where Mtt is 4-methyltrityl, is used for the lysine at position 17 and Boc-Tyr(t-Bu)-OH is used for the tyrosine at position 1.
  • Fmoc groups are removed prior to each coupling step (2 ⁇ 10 minutes) using 20% piperidine in DMF.
  • the Mtt protecting group on the lysine at position 17 is selectively removed from the peptide resin using three treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (3 ⁇ 20-minute treatment), and the resin is thoroughly washed with DCM and DMF.
  • mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China) is coupled for 1 hour using a 4-fold excess of the diacid, PyBOP, and diisopropylethylamine (1:1:1 mol/mol/mol) in 1:1 DCM/DMF.
  • the peptide-resin is washed with DCM and then thoroughly dried over vacuum.
  • the dry peptide-resin is treated with cleavage cocktail (10 mL TFA, 0.5 mL triisopropylsilane, 0.5 mL water, and 0.5 mL 1,2-ethanedithiol) for 2 hours at room temperature.
  • the peptide resin solution is filtered into a 50-mL conical centrifuge tube and treated with 5-fold excess volume of cold diethyl ether ( ⁇ 20° C.) to precipitate the crude peptide.
  • the peptide/ether suspension is centrifuged at 3000 rcf for 1.5 min. to form a solid pellet and the supernatant is decanted.
  • the pellet is washed further two times with cold diethyl ether, centrifuging for 1 min. each time, then dried in vacuo.
  • the crude peptide is solubilized in 20% acetic acid/80% water and purified by RP-HPLC on a SymmetryPrep 7 ⁇ m C18 preparative column (18 ⁇ 300 mm, Waters) with linear gradients of 100% acetonitrile and 0.1% TFA/water buffer system (25-45% acetonitrile in 65 min).
  • the purity of peptide is assessed using analytical RP-HPLC and pooling criteria is >95%.
  • the main pool purity of compound of example 1 is found to be 96.8%.
  • SEQ ID NO:6 The structure of SEQ ID NO:6 is depicted below using the standard single letter amino acid codes with the exceptions of residues D-Ala2, K17, Aib20, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:6 is prepared substantially as described by the procedures of Example 1, where instead the protected diacid is mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China).
  • SEQ ID NO:7 The structure of SEQ ID NO:7 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, ⁇ MeL13, K17, Aib20, 4-Pal25, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:7 is prepared substantially as described by the procedures of Example 1.
  • SEQ ID NO:8 The structure of SEQ ID NO:8 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, ⁇ MeL13, Orn16, K17, Aib20, 4-Pal25, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:8 is prepared substantially as described by the procedures of Example 1.
  • SEQ ID NO:9 The structure of SEQ ID NO:9 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, ⁇ MeL13, Aib20, K26, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:9 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17.
  • SEQ ID NO:10 The structure of SEQ ID NO:10 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, ⁇ MeL13, Orn16, Aib20, K26, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:10 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17.
  • SEQ ID NO:11 The structure of SEQ ID NO:11 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, ⁇ MeL13, Orn16, Aib20, D-Glu24, K26, and Ser39, where the structures of these amino acid residues have been expanded:
  • the compound according to SEQ ID NO:11 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17 and the protected diacid is mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China).
  • Example 8 SEQ ID NO:12
  • Example 122 SEQ ID NO:126
  • Glucagon (referred to as Gcg or hGcg) is a Reference Standard prepared at Eli Lilly and Company.
  • GLP-1(7-36)-NH 2 (referred to as GLP-1 or hGLP-1) is obtained from CPC Scientific (Sunnyvale, CA, 97.2% purity, 100 ⁇ M aliquots in 100% DMSO).
  • GIP(1-42)-NH 2 (referred to as GIP) is prepared at Lilly Research Laboratories using peptide synthesis and HPLC chromatography as described above (>80% purity, 100 ⁇ M aliquots in 100% DMSO).
  • [ 125 I]-radiolabeled Gcg, GLP-1, or GIP is prepared using [ 125 I]-lactoperoxidase and obtained from Perkin Elmer (Boston, MA).
  • Stably transfected cell lines are prepared by subcloning receptor cDNA into a pcDNA3 expression plasmid and transfected into human embryonic kidney (HEK) 293 (hGcgR and hGLP-1R) or Chinese Hamster Ovary (CHO) (hGIPR) cells followed by selection with Geneticin (hGLP-1R and hGIPR) or hygromycin B (hGcgR).
  • HEK human embryonic kidney
  • hGcgR and hGLP-1R human embryonic kidney
  • hGIPR Chinese Hamster Ovary cells
  • Method 1 Frozen cell pellets are lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5, and Roche CompleteTM Protease Inhibitors with EDTA.
  • the cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes.
  • the homogenate is centrifuged at 4° C. at 1100 ⁇ g for 10 minutes.
  • the supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer and rehomogenized as described above.
  • the homogenate is centrifuged at 1100 ⁇ g for 10 minutes.
  • the second supernatant is combined with the first supernatant and centrifuged at 35000 ⁇ g for 1 hour at 4° C.
  • the resulting membrane pellet is resuspended in homogenization buffer containing protease inhibitors at approximately 1 to 3 mg/mL, quick frozen in liquid nitrogen and stored as aliquots in a ⁇ 80° C. freezer until use.
  • Method 2 Frozen cell pellets are lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5, 1 mM MgCl 2 , Roche CompleteTM EDTA-free Protease Inhibitors and 25 units/mL DNAse I (Invitrogen).
  • the cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 20 to 25 strokes.
  • the homogenate is centrifuged at 4° C. at 1800 ⁇ g for 15 minutes. The supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer (without DNAse I) and rehomogenized as described above.
  • the homogenate is centrifuged at 1800 ⁇ g for 15 minutes.
  • the second supernatant is combined with the first supernatant and centrifuged an additional time at 1800 ⁇ g for 15 minutes.
  • the overall supernatant is then centrifuged at 25000 ⁇ g for 30 minutes at 4° C.
  • the resulting membrane pellet is resuspended in homogenization buffer (without DNAse I) containing protease inhibitors at approximately 1 to 3 mg/mL and stored as aliquots in a ⁇ 80° C. freezer until use.
  • the equilibrium binding dissociation constants (K d ) for the various receptor/radioligand interactions are determined from homologous competition binding analysis instead of saturation binding due to high propanol content in the [ 125 I] stock material.
  • the K d values determined for the receptor preparations were as follows: hGcgR (3.9 nM), hGLP-1R (1.2 nM) and hGIPR (0.14 nM).
  • the human Gcg receptor binding assays are performed using a Scintillation Proximity Assay (SPA) format with wheat germ agglutinin (WGA) beads (Perkin Elmer).
  • the binding buffer contains 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 0.1% (w/v) bacitracin (Research Products), 0.003% (w/v) Polyoxyethylenesorbitan monolaurate (TWEEN®-20), and Roche CompleteTM Protease Inhibitors without EDTA.
  • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • Peptides and Gcg are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves).
  • 5 ⁇ L serially diluted compound or DMSO is transferred into Corning® 3632 clear bottom assay plates containing 45 ⁇ L assay binding buffer or unlabeled Gcg control (non-specific binding or NSB, at 1 ⁇ M final).
  • 50 ⁇ L human GcgR membranes 1.5 ⁇ g/well
  • 50 ⁇ L of WGA SPA beads 80 to 150 ⁇ g/well
  • Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 12 hours of incubation/settling time at room temperature.
  • Final assay concentration ranges for peptides tested in response curves is typically 1150 nM to 0.058 nM and for the control Gcg from 1000 nM to 0.05 nM.
  • the human GLP-1 receptor binding assay is performed using an SPA format with WGA beads.
  • the binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, and Roche CompleteTM Protease Inhibitors without EDTA.
  • Peptides and GLP-1 are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves).
  • Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 5 to 12 hours of incubation/settling time at room temperature.
  • Final assay concentration ranges for peptides tested in response curves are typically 1150 nM to 0.058 nM and for the control GLP-1, 250 nM to 0.013 nM.
  • the human GIP receptor binding assay is performed using an SPA format with WGA beads.
  • the binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, and Roche CompleteTM Protease Inhibitors without EDTA.
  • Peptides and GIP are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves).
  • Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 2.5 to 12 hours of incubation/settling time at room temperature.
  • Final assay concentration ranges for peptides tested in response curves is typically 1150 to 0.058 nM or 115 nM to 0.0058 nM and for the control GIP, 250 nM to 0.013 nM.
  • Raw CPM data for concentration curves of peptides, Gcg, GLP-1, or GIP are converted to percent inhibition by subtracting nonspecific binding (binding in the presence of excess unlabeled Gcg, GLP-1, or GIP, respectively) from the individual CPM values and dividing by the total binding signal, also corrected by subtracting nonspecific binding.
  • Data are analyzed using four-parameter (curve maximum, curve minimum, IC 50 , Hill slope) nonlinear regression routines (Genedata Screener, version 12.0.4, Genedata AG, Basal, Switzerland).
  • examples of the present invention are very potent binders of the human GIPR, and have lower affinity for the GLP-1R and GcgR.
  • a set of cAMP assays are conducted in HEK293 cells expressing the human GLP-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR), or glucagon receptor (GcgR).
  • GLP-1R human GLP-1 receptor
  • GIPR glucose-dependent insulinotropic peptide receptor
  • GcgR glucagon receptor
  • Each receptor over-expressing cell line (20 ⁇ l) is treated with the test peptide in DMEM (Gibco Cat #31053) supplemented with 0.1% Casein (Sigma Cat #C4765), 250 ⁇ M IBMX, 1 ⁇ GlutaMAXTM (Gibco Cat #35050), and 20 mM HEPES (HyClone Cat #SH30237.01) in a 20 ⁇ l assay volume.
  • cAMP Dynamic 2 HTRF Assay Kit 62AM4PEJ
  • the Lysis buffer containing cAMP-d2 conjugate (20 ⁇ l) and the antibody anti-cAMP-Eu3+-Cryptate (20 ⁇ l) are then added to determine the cAMP level.
  • HTRF signal is detected with an Envision 2104 plate reader (PerkinElmer). Fluorescent emission at 620 nm and at 665 nm is measured and the ratio between 620 nm and at 665 nm is calculated and then are converted to nM cAMP per well using a cAMP standard curve.
  • Dose response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four parameter non-linear regression fit with a variable slope (Genedata Screener 13).
  • EC50 is the concentration of compound causing half-maximal simulation in a dose response curve.
  • a relative EC 50 value is derived by non-linear regression analysis using the percent maximal response vs. the concentration of peptide added, fitted to a four-parameter logistic equation.
  • Example and comparator molecules are conducted to determine the intrinsic potency of Example and comparator molecules performed in the presence of casein (instead of serum albumin) as a nonspecific blocker, which does not interact with the fatty acid moieties of the analyzed molecules.
  • Intracellular cAMP levels are determined by extrapolation using a standard curve. Dose response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four-parameter non-linear regression fit with a variable slope (Genedata Screener 13). EC 50 is the concentration of compound causing half-maximal simulation in a dose response curve. Each relative EC50 value for the geometric mean calculation is determined from a curve fitting.
  • Concentration response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four-parameter non-linear regression fit with a variable slope (Genedata Screener 13).
  • EC50 is the concentration of compound causing half-maximal simulation in a dose response curve.
  • the EC 50 summary statistics are computed as follows: Geometric mean:
  • test peptides are run plus the native ligands GIP, GLP-1, and glucagon, buffer only as baseline (minimum) and the highest concentration of the respective GIP, GLP-1, and glucagon standard is used as maximum for calculations.
  • the peptide is tested in 8 runs of the hGIPR cAMP assay.
  • hGIP amide, hGLP-1 amide, and glucagon EC50 in Table 2 are illustrative of geometric mean values from a series of 18 assay values, and values will vary each day compared to the zero buffer. Accordingly, each Example will use the geometric mean of those values to normalize the Example assay runs.
  • Example compounds of the present invention are very potent stimulating cAMP from human GIPR in the presence of 0.1% casein.
  • the pharmacokinetics of select Examples are evaluated following a single subcutaneous administration of 50 nmol/kg to male cynomolgus monkeys. Blood samples are collected over 504 hours and resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K 3 EDTA) concentrations are determined using a qualified LC/MS method that measured the intact mass of the compound. Each peptide and an analog as an internal standard are extracted from 100% cynomolgus monkey plasma using a protein precipitation method. Instruments are combined for LC/MS detection. Mean pharmacokinetic parameters are shown in Table 3.
  • the pharmacokinetics of select Examples are evaluated following a single subcutaneous (SC) administration of 100 nmol/kg to male Sprague Dawley rats. Blood samples are collected over 168 hours following SC administration. Pharmacokinetic parameters are calculated using individual plasma concentrations. A qualified LC/MS method that measures the intact mass of the Example is used to determine plasma (K 3 EDTA) concentrations. Each peptide and an analog as an internal standard are extracted from 100% rat plasma using a protein precipitation method. Instruments are combined for LC/MS detection. Mean pharmacokinetic parameters for the Examples are shown in Table 4.
  • Rats with femoral artery and femoral vein canulas (Envigo, Indianapolis, IN) (280-320 grams) are single-housed in polycarbonate cages with filter tops. Rats maintained on a 12:12 h light-dark cycle (lights on at 6:00 A.M.) at 21° C. and receive food and deionized water ad libitum. Rats are randomized by body weight and dosed 1.5 mL/kg subcutaneously (s.c.) at doses of 0.3, 1.0, 3, 10, 30, and 100 nmol/kg 16 hours prior to glucose administration then fasted.
  • mice are weighed and anesthetized with sodium pentobarbital dosed intraperitoneally (i.p.) (65 mg/kg, 30 mg/mL).
  • a blood sample is collected into EDTA tubes after which glucose is administered intravenously (i.v.) (0.5 mg/kg, 5 mL/kg).
  • Blood samples are collected for glucose and insulin levels at 2, 4, 6, 10, 20 and 30 min post-intravenous administration of glucose.
  • Plasma glucose levels are determined using a clinical chemistry analyzer.
  • Insulin secretion ivGTT
  • SEM Insulin secretion
  • CD4+ T Cell Assay The CD4+ T cell assay is used to compare the compounds of Examples 1, 2, 3, 5, and 6 for a potential to induce an immune response in vivo according to methods known in the art (see, e.g., Jones et al. (2004) J. Interferon Cytokine Res. 24:560-572; and Jones et al. (2005) J. Thromb. Haemost. 3:991-1000), where an assessment of clinically tested monoclonal antibodies and peptides shows some degree of correlation between T cell proliferation observed in vitro and immunogenicity in the clinic. Protein therapeutics that induce less than 30% positive response in the CD4+ T cell proliferation assay are associated with a low risk of immunogenicity.
  • T cell-depleted peripheral blood mononuclear cells are prepared and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) from a cohort of 10 healthy donors with diverse human leukocyte antigen (HLA) class II haplotypes. Each donor is tested in triplicate with 2.0 mL media control, keyhole limpet hemocyanin (KLH; 0.33 ⁇ M), and the compounds of Examples 1, 2, 3, 5, and 6 (0.33 ⁇ M). Cultures are incubated for 7 days at 37° C. with 5% CO 2 .
  • CSE carboxyfluorescein diacetate succinimidyl ester
  • samples are analyzed by flow cytometry using a BD LSR II Fortessa (Becton Dickinson; Franklin Lakes, NJ), equipped with a high throughput sampler (HTS). Data is analyzed using FlowJo® Software (FlowJo, LLC/TreeStar; Ashland, OR).
  • GIP 1-42 (Human) SEQ ID NO: 1 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP-1 (7-36) amide (Human) SEQ ID NO: 2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 Glucagon (Human) SEQ ID NO: 3 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO: 4 Z 1 X 1 X 2 EGTX 6 ISDYSIX 13 LDX 16 X 17 X 18 QX 20 X 21 X 22 VX 24 X 25 X 26 LX 28 X 29 GPSSGAPPPSZ 2

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Abstract

The present invention relates to compounds having activity at the human glucose-dependent insulinotropic polypeptide (GIP) receptor. The present invention also relates to compounds having an extended duration of action at the GIP receptor. Such compounds may be useful in the treatment of diabetes, including type 2 diabetes mellitus (“T2DM”). Also, the compounds may be useful in the treatment of obesity.

Description

The present invention relates to compounds having activity at the human glucose-dependent insulinotropic polypeptide (GIP) receptor. The present invention also relates to compounds having an extended duration of action at the GIP receptor. Compounds may be useful in the treatment of type 2 diabetes mellitus (“T2DM”). Also, the compounds may be useful in the treatment of obesity.
Over the past several decades, the prevalence of diabetes has continued to rise. T2DM is the most common form of diabetes, accounting for approximately 90% of all diabetes. T2DM is characterized by high blood glucose levels associated mainly with insulin resistance. The current standard of care for T2DM includes diet and exercise, treatment with oral medications, and injectable glucose-lowering drugs, including incretin-based therapies such as GLP-1 receptor agonists. A variety of GLP-1 receptor agonists are currently available for treatment of T2DM, although currently marketed GLP-1 receptor agonists are generally dose-limited by gastrointestinal side effects such as nausea and vomiting.
Subcutaneous injection is the typical route of administration for the available GLP-1 receptor agonists. When treatment with oral medications and incretin-based therapies are insufficient, insulin treatment is considered. Despite the advances in treatment available today, many patients with T2DM are unable to reach their glycemic control goals. Uncontrolled diabetes leads to several conditions associated with increased morbidity and mortality of patients.
There is a need for a treatment to enable more patients with T2DM to reach their glycemic treatment goals.
Obesity is a complex medical disorder resulting in excessive accumulation of adipose tissue mass. Today obesity is a global public health concern that is associated with undesired health outcomes and morbidities. Desired treatments for patients with obesity should reduce excess body weight, improve obesity-related co-morbidities, and maintain long-term weight reduction. Available treatments for obesity are particularly unsatisfactory for patients with severe obesity. There is a need for alternative treatment options to induce therapeutic weight loss in patients in need of such treatment.
WO2016/111971 describes peptides stated to have GLP-1R and GIPR agonist activities. WO2013/164483 also discloses compounds stated to have GLP-1R and GIPR activities.
WO2018/181864 discloses compounds stated to have GIPR agonist activity.
There is a need for T2DM treatments capable of providing effective glucose control for a larger portion of the patients in need of such treatment. There is a further need for T2DM treatments capable of providing effective glucose control and with a favorable side effect profile. There is a need for alternate treatment options to provide therapeutic weight loss in a patient in need of such treatment, such as a patient with severe obesity. There is a desire for diabetes treatment options that may be combined with insulin therapy and/or incretin therapy to provide the patient with superior glycemic outcomes and/or more desirable side effect profiles.
Compounds with extended duration of action at the GIP receptor are desirable to allow for less frequent dosing of the compound.
Accordingly, embodiment 1 is a compound of Formula I
Z1X1X2EGTX6ISDYSIX13LDX16X17X18QX20X21X22VX24X25X26L X28X29GPSSGAPPPSZ2 (SEQ ID NO:4) wherein:
    • Z1 is a modification of the N-terminal amino group wherein the modification is selected from the group consisting of acetyl and absent;
    • X1 is selected from the group consisting of Y and D-Tyr;
    • X2 is selected from the group consisting of Aib, A, and D-Ala;
    • X6 is selected from the group consisting of F, αMeF, Iva, L, αMeL, and αMeF(2F);
    • X13 is selected from the group consisting of αMeL, A, L, and Aib;
    • X16 is selected from the group consisting of K, E, and Orn;
    • X17 is selected from the group consisting of I and K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H;
    • X18 is selected from the group consisting of H, A, and R;
    • X20 is selected from the group consisting of Aib and Q;
    • X21 is selected from the group consisting of D and E;
    • X22 is selected from the group consisting of F and αMeF;
    • X24 is selected from the group consisting of E, N, Q, and D-Glu;
    • X25 is selected from the group consisting of Y, 4-Pal, W, and αMeY;
    • X26 is selected from the group consisting of L and K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H;
    • X28 is selected from the group consisting of E and A;
    • X29 is selected from the group consisting of G, A, Q, and T; q is selected from the group consisting of 16 and 18; and
    • Z2 is absent or a modification of the C-terminal group, wherein the modification is amidation;
    • wherein one and only one selected from X17 and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H;
      or a pharmaceutically acceptable salt thereof.
An embodiment 2 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein Z1 is absent and X1 is Y.
An embodiment 3 provides a compound, or pharmaceutically acceptable salt thereof, of embodiment 1 or embodiment 2 wherein X2 is Aib.
An embodiment 4 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 3 wherein:
X6 is selected from the group consisting of F and αMeF(2F).
An embodiment 5 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 4 wherein:
X13 is selected from the group consisting of L and αMeL.
An embodiment 6 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 5 wherein X16 is K or Orn.
An embodiment 7 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 6 wherein X16 is K.
An embodiment 8 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 7 wherein X18 is H.
An embodiment 9 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 8 wherein X20 is Aib; and X22 is F.
An embodiment 10 provides a compound, or a pharmaceutically acceptable salt thereof, as claimed of any one of embodiments 1 to 9 wherein X21 is D.
An embodiment 11 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 10 wherein X25 is 4-Pal or Y.
An embodiment 12 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 11 wherein:
    • X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H; and
    • X26 is L.
An embodiment 13 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 11 wherein:
    • X17 is I; and
    • X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H.
An embodiment 14 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 13 wherein q is 16.
An embodiment 15 provides a compound, or a pharmaceutically acceptable salt thereof, of any one of embodiments 1 to 13 wherein q is 18.
An embodiment 16 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein: Z1 is absent;
    • X1 is Y;
    • X2 is selected from the group consisting of Aib and D-Ala; X6 is F;
    • X13 is selected from the group consisting of αMeL and L;
    • X16 is selected from the group consisting of K and Orn;
    • X18 is selected from the group consisting of H and A;
    • X20 is Aib;
    • X22 is F;
    • X24 is selected from the group consisting of E, N, and D-Glu;
    • X25 is selected from the group consisting of Y, 4-Pal, and W;
    • X28 is selected from the group consisting of E and A;
    • X29 is selected from the group consisting of G, A, and Q; and q is selected from the group consisting of 16 and 18.
An embodiment 17 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 16 wherein:
    • Z1 is absent;
    • X2 is Aib;
    • X13 is αMeL;
    • X18 is H;
    • X24 is selected from the group consisting of E and D-Glu;
    • X25 is selected from the group consisting of Y and 4-Pal;
    • X28 is E;
    • X29 is selected from the group consisting of G and A.
An embodiment 18 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 17 wherein: X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H; and
X26 is L.
An embodiment 19 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 17 wherein: X17 is I; and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)q—CO2H.
An embodiment 20 provides a compound, or a pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
An embodiment 21 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:5. An embodiment 22 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:6. An embodiment 23 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:7. An embodiment 24 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:8. An embodiment 25 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:9. An embodiment 26 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ ID NO:10. An embodiment 27 provides a compound or pharmaceutically acceptable salt thereof, of embodiment 1 wherein the compound is SEQ NO:11.
An embodiment provides a method of treating a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome, comprising administering to a subject in need thereof, an effective amount of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof. An embodiment provides a method of treating a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome, comprising administering to a subject in need thereof, an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof. An embodiment provides a method for providing therapeutic weight loss comprising administering to a subject in need thereof, an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. An embodiment is a method of treating T2DM comprising administering to a subject in need thereof, and effective amount of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof.
An embodiment provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy. An embodiment provides a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, for use in therapy to treat a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome. An embodiment provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in therapy to treat a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome. In an embodiment, the condition is T2DM. In an embodiment, the condition is obesity. In an embodiment, the condition is type 1 diabetes (T1DM). In an embodiment, the condition is diabetes in a patient receiving insulin therapy. In an embodiment, the condition is metabolic syndrome.
The compounds of Formula I, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of a variety of symptoms or disorders. For example, certain embodiments provide a method for treatment of T2DM in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In an embodiment is a method for treatment of obesity in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In an embodiment, the method is inducing non-therapeutic weight loss in a subject, comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
In certain embodiments, the present invention provides a method for treatment of metabolic syndrome in a patient comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. In an embodiment, the method is treatment of diabetes in a patient receiving insulin treatment, comprising administering to a subject in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. Also provided herein is a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, a growth differentiation factor 15 modulator (“GDF15”), a peptide tyrosine tyrosine modulator (“PYY”), a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and an oxyntomodulin agonist (“OXM”) in the treatment of a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome. Also provided herein is a compound of the present invention for use in simultaneous, separate, and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, a growth differentiation factor 15 modulator (“GDF15”), a peptide tyrosine tyrosine analog (“PYY”), a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1-glucagon receptor agonist including oxyntomodulin and analogs thereof, in the treatment of a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome. In an embodiment, a compound of the present invention is provided in a fixed dose combination with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM. In an embodiment, a compound of the present invention is provided in a fixed dose combination with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1-glucagon receptor agonist including OXM and analogs thereof. In an embodiment is a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, a dual amylin-calcitonin receptor agonist, and OXM in the treatment of a condition selected from the group consisting of T2DM and obesity. In an embodiment is a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1 glucagon receptor agonist including OXM and analogs of, in the treatment of a condition selected from the group consisting of T2DM and obesity. In an embodiment is a compound of the present invention for use in simultaneous, separate and sequential combinations with one or more agents selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, and a SGLT-2 inhibitor in the treatment of a condition selected from the group consisting of T2DM and obesity.
In an embodiment is a method for treating diabetes in a patient receiving insulin therapy, comprising administering an effective amount of a Compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient in need thereof. An embodiment is treatment to a patient administered insulin therapy for T1DM. An embodiment is treatment to patient administered insulin therapy for T2DM. An embodiment is once weekly dosing. An embodiment is subcutaneous treatment once weekly to a patient administered insulin therapy. An embodiment exists wherein the insulin therapy comprises basal insulin therapy. An embodiment exists wherein the insulin therapy comprises mealtime insulin therapy. An embodiment exists wherein the insulin therapy comprises ultra-rapid insulin therapy. Insulin therapy administered with acute infusions of a compound of Formula I, or a pharmaceutically acceptable salt thereof, may enhance glucagon excursion in patients undergoing hypoglycemic clamp, thus enhancing the body's natural defense against hypoglycemia. A compound of Formula I, or a pharmaceutically acceptable salt thereof, can be dosed once weekly independent of the type of insulin used or doses of insulin used. An embodiment is a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, administered as an effective amount to a patient receiving insulin therapy, independent of the type of insulin used or doses of insulin used. An embodiment is a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, dosed once weekly as an effective amount to a patient receiving insulin therapy independent of the type of insulin used or doses of insulin used. As used herein “insulin therapy” means treatment of a patient with diabetes using an approved insulin treatment. Such insulin therapy is known to the skilled artisan and/or clinical health care professional. For example, insulin therapy may comprise treatment using basal insulin. Such basal insulin “insulin therapy” may be used in a dosing regimen with mealtime insulin and/or ultra-rapid insulin. As used herein, “mealtime insulin” means insulin and/or modified insulin to be administered with meals, for example, but not limited to, insulin lispro. As used herein, “basal insulin” means modified insulin with a longer duration of action, such as, for example, but not limited to, insulin glargine.
Another embodiment provides the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a condition selected from the group consisting of T2DM, obesity, and metabolic syndrome. An embodiment provides the use of a compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a condition selected from the group consisting of diabetes, obesity, and metabolic syndrome. In an embodiment, the medicament is for the treatment of T2DM. In an embodiment, the medicament is for the treatment of obesity. In an embodiment, the medicament is for use in the treatment of diabetes in a patient receiving insulin therapy.
Another embodiment provides a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and at least one selected from the group consisting of a carrier, diluent, and excipient. In an embodiment, a pharmaceutical composition for subcutaneous administration is provided.
As used herein, the term “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of a symptom, condition, or disorder.
Certain compounds of the present invention are generally effective over a wide dosage range. For example, dosages for once weekly parenteral dosing may fall within the range of 0.05 mg to about 60 mg per person per week.
The compounds of Formula I, or a pharmaceutically acceptable salt thereof, include novel amino acid sequences having affinity for the GIP receptor, with desired potency at the receptor. GIP is a 42 amino acid peptide (SEQ ID NO:1), which, like GLP-1, is known as an incretin, and it plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose.
GLP-1 is a 36 amino acid peptide, the major biologically active fragment of which is produced as a 30-amino acid, C-terminal amidated peptide (GLP-17-36) (SEQ ID NO:2).
Glucagon is a 29-amino acid peptide hormone (SEQ ID NO:3) secreted by α-cells of the islet of Langerhans in the pancreas and is involved in glucose homeostasis.
The compounds of present invention provide desired potency at the GIP receptor with high degree of selectivity against the GLP-1R and the Glucagon receptor. In an embodiment, compounds have desired GIP receptor activity with extended duration of action.
As used herein the term “amino acid” means both naturally occurring amino acids and unnatural amino acids. The amino acids are typically depicted using standard one letter codes (e.g., L=leucine), as well as alpha-methyl substituted residues of natural amino acids (e.g., α-methyl leucine, or αMeL, and α-methyl phenylalanine, or αMeF) and certain other unnatural amino acids, such as alpha-amino isobutyric acid, or “Aib,” “4Pal,” “Orn,” and the like. The structures of these amino acids appear below:
Figure US11897926-20240213-C00001
As used herein, “Orn” means L-ornithine. As used herein, “4Pal” means 3-(4-Pyridyl)-L-alanine. As used herein, “αMeF(2F)” means alpha-methyl 2-fluoro-L-phenylalanine. As used herein, “αMeY” and “αMeL” mean alpha-methyl-L-tyrosine and alpha-methyl-L-leucine, respectively. As used herein, “e” and “D-Glu” mean D-glutamic acid. As used herein, “D-Tyr” and “y” each mean D-tyrosine. As used herein, “D-Ala” and “a” each mean D-alanine. As used herein, “αMeF” means alpha-methyl-F and alpha-methyl-Phe. As used herein “Iva” means L-isovaline.
In an embodiment, the conjugation is an acylation. In an embodiment, the conjugation is to the epsilon-amino group of the K side-chain. In an embodiment of the compounds of the present invention, a fatty acid moiety is conjugated, via a linker, to a K at position 17. In an embodiment of the compounds of the present invention, a fatty acid moiety is conjugated, via a linker, to a K at position 26.
In an embodiment, q is selected from the group consisting of 16 and 18. In an embodiment, q is 16. In an embodiment, q is 18.
When used herein in reference to the GIP receptor the terms “activity,” “activate[s]” “activat[ing]” and the like refers to the capacity of a compound, or a pharmaceutically acceptable salt thereof, to bind to and induce a response at the receptor, as measured using assays known in the art, such as the in vitro assays described below.
The affinity of compounds of Formula I, or a pharmaceutically acceptable salt thereof, for the GIP receptor may be measured using techniques known for measuring receptor binding levels in the art, including, for example, those described in the examples below, and is commonly expressed as a Ki value. The activity of the compounds of the present invention at the GIP receptor may also be measured using techniques known in the art, including for example the in vitro activity assays described below, and is commonly expressed as an EC50 value, which is the concentration of compound causing half-maximal simulation in a dose response curve.
In addition, data is provided for each compound for activity and affinity at the GLP-1 and glucagon receptors, to demonstrate the degree of selectivity of the compounds of the present invention for the GIPR.
In an embodiment, a pharmaceutical composition of a compound of Formula I is suitable for administration by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Some pharmaceutical compositions and processes for preparing same are well known in the art, (See, e.g., Remington: The Science and Practice of Pharmacy (D. B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006)).
Compounds of the present invention may react with any of a number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. (See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts of the present invention include, but are not limited to, sodium, trifluoroacetate, hydrochloride, ammonium, and acetate salts. In an embodiment, a pharmaceutically acceptable salt is selected from the group consisting of sodium, hydrochloride, and acetate salts.
The present invention also encompasses novel intermediates and processes useful for the synthesis of compounds of the present invention, or a pharmaceutically acceptable salt thereof. The intermediates and compounds of the present invention may be prepared by a variety of procedures known in the art. In particular, the Examples below describe a process using chemical synthesis. The specific synthetic steps for each of the routes described may be combined in different ways to prepare compounds of the present invention. The reagents and starting materials are readily available to one of ordinary skill in the art.
When used herein, the term “effective amount” refers to the amount or dose of a compound of the present invention, or a pharmaceutically acceptable salt thereof, which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment. An effective amount can be determined by a person of skill in the art using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a subject, a number of factors are considered, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
When used herein, the term “subject in need thereof” refers to a mammal, preferably a human, with a disease or condition requiting treatment or therapy, including for example those listed in the preceding paragraphs.
As used herein, “EDTA” means ethylenediaminetetraacetic acid. As used herein, “DMSO” means dimethyl sulfoxide. As used herein, “CPM” means counts per minute. As used herein, “IBMX” means 3-isobutyl-1-methylxanthine. As used herein, “LC/MS” means liquid chromatography/mass spectrometry. As used herein, “HTRF” means homogeneous time-resolved fluorescence. As used herein, “DMF” refers to N,N-dimethylformamide. As used herein, “DCM” refers to dichloromethane. As used herein, “TFA” refers to trifluoroacetic acid. As used herein, “TFA salt” refers to trifluoroacetate salt. As used herein, “RP-HPLC” means reversed-phase high performance liquid chromatography.
The invention is further illustrated by the following examples, which are not to be construed as limiting.
Peptide Synthesis EXAMPLE 1 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)18—CO2H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH2 (SEQ ID NO:5)
The structure of SEQ ID NO:5 is depicted below using the standard single letter amino acid codes with the exceptions of residues D-Ala2, K17, Aib20, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00002
The peptide backbone of Example 1 is synthesized using fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl (t-Bu) chemistry on a Symphony multiplex peptide synthesizer (Gyros Protein Technologies. Tucson, AZ; 3.3.0.1), software version 3.3.0.
The resin consists of aminomethyl polystyrene functionalized with a Rink Amide linker (polystyrene AM RAM, RAPP polymeric GmbH, H40023, 200-400 mesh) at a substitution of 0.8 mmol/g. Standard side-chain protecting groups are used with the following exceptions: Fmoc-Lys(Mtt)-OH, where Mtt is 4-methyltrityl, is used for the lysine at position 17 and Boc-Tyr(t-Bu)-OH is used for the tyrosine at position 1. Fmoc groups are removed prior to each coupling step (2×10 minutes) using 20% piperidine in DMF. All standard amino acid couplings are performed using an equal molar ratio of Fmoc amino acid (0.3 M in DMF), diisopropylcarbodiimide (0.9 M in DCM) and Oxyma (0.9 M in DMF) at a 9-fold molar excess over the theoretical peptide loading. Couplings are allowed to proceed for 1.5 hours, with the following exceptions: coupling of valine, 3 hours; coupling of Cα-methylated amino acids, 6 hours; coupling to Cα-methylated amino acids, 6-10 hours. After completion of the synthesis of the peptide backbone, the resin is thoroughly washed with DCM to remove residual DMF. The Mtt protecting group on the lysine at position 17 is selectively removed from the peptide resin using three treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (3×20-minute treatment), and the resin is thoroughly washed with DCM and DMF.
Subsequent attachment of the fatty acid-linker moiety is accomplished by coupling of 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.) and Fmoc-glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.) following the procedures described above for standard coupling and deprotection reactions. After removal of the final Fmoc protecting group, mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China) is coupled for 1 hour using a 4-fold excess of the diacid, PyBOP, and diisopropylethylamine (1:1:1 mol/mol/mol) in 1:1 DCM/DMF.
After the synthesis is complete, the peptide-resin is washed with DCM and then thoroughly dried over vacuum. The dry peptide-resin is treated with cleavage cocktail (10 mL TFA, 0.5 mL triisopropylsilane, 0.5 mL water, and 0.5 mL 1,2-ethanedithiol) for 2 hours at room temperature. The peptide resin solution is filtered into a 50-mL conical centrifuge tube and treated with 5-fold excess volume of cold diethyl ether (−20° C.) to precipitate the crude peptide. The peptide/ether suspension is centrifuged at 3000 rcf for 1.5 min. to form a solid pellet and the supernatant is decanted. The pellet is washed further two times with cold diethyl ether, centrifuging for 1 min. each time, then dried in vacuo. The crude peptide is solubilized in 20% acetic acid/80% water and purified by RP-HPLC on a SymmetryPrep 7 μm C18 preparative column (18×300 mm, Waters) with linear gradients of 100% acetonitrile and 0.1% TFA/water buffer system (25-45% acetonitrile in 65 min). The purity of peptide is assessed using analytical RP-HPLC and pooling criteria is >95%. The main pool purity of compound of example 1 is found to be 96.8%. Subsequent lyophilization of the final main product pool yielded the lyophilized peptide TFA salt. The molecular weight is determined by LC/MS (Found: [M+3H]3+=1638.4; Calculated [M+3H]3+=1638.53; Found MW (avg)=4912.2; Calc. MW (avg): 4912.58).
EXAMPLE 2 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)16—CO2H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH2 (SEQ ID NO:6)
The structure of SEQ ID NO:6 is depicted below using the standard single letter amino acid codes with the exceptions of residues D-Ala2, K17, Aib20, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00003
The compound according to SEQ ID NO:6 is prepared substantially as described by the procedures of Example 1, where instead the protected diacid is mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China). The molecular weight is determined by LC/MS (Found: [M+3H]3+=1629.15; Calc. [M+3H]3+=1629.18; Found MW (avg)=4884.45; Calc. MW (avg)=4884.53).
EXAMPLE 3 Y-Aib-EGTFISDYSI-αMeL-LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)18—CO2H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH2 (SEQ ID NO:7)
The structure of SEQ ID NO:7 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, αMeL13, K17, Aib20, 4-Pal25, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00004
The compound according to SEQ ID NO:7 is prepared substantially as described by the procedures of Example 1. The molecular weight is determined by LC/MS (Found: [M+3H]3+=1662.52; Calc. [M+3H]3+=1662.55; Found MW (avg)=4984.55; Calc. MW (avg)=4984.64).
EXAMPLE 4 Y-Aib-EGTFISDYSI-αMeL-LD-Orn-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)18—CO2H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH2 (SEQ ID NO:8)
The structure of SEQ ID NO:8 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, αMeL13, Orn16, K17, Aib20, 4-Pal25, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00005
The compound according to SEQ ID NO:8 is prepared substantially as described by the procedures of Example 1. The molecular weight is determined by LC/MS (Found: [M+3H]3+=1657.82; Calc. [M+3H]3+=1657.87; Found MW (avg)=4970.46; Calc. MW (avg)=4970.62).
EXAMPLE 5 Y-Aib-EGTFISDYSI-αMeL-LDKIHQ-Aib-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)18—CO2H)LEGGPSSGAPPPS-NH2 (SEQ ID NO:9)
The structure of SEQ ID NO:9 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, αMeL13, Aib20, K26, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00006
The compound according to SEQ ID NO:9 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17. The molecular weight is determined by LC/MS (Found: [M+3H]3+=1662.8; Calc. [M+3H]3+=1662.88; Found MW (avg)=4985.4; Calc. MW (avg)=4985.63).
EXAMPLE 6 Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)18—CO2H)LEGGPSSGAPPPS-NH2 (SEQ ID NO:10)
The structure of SEQ ID NO:10 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, αMeL13, Orn16, Aib20, K26, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00007
The compound according to SEQ ID NO:10 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17. The molecular weight is determined by LC/MS (Found: [M+3H]3+=1658.1; Calc. [M+3H]3+=1658.20; Found MW (avg)=4971.3; Calc. MW (avg)=4971.6).
EXAMPLE 7 Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-EFV-(D-Glu)-YK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO—(CH2)16—CO2H)LEGGPSSGAPPPS-NH2 (SEQ ID NO:11)
The structure of SEQ ID NO:11 is depicted below using the standard single letter amino acid codes with the exceptions of residues Aib2, αMeL13, Orn16, Aib20, D-Glu24, K26, and Ser39, where the structures of these amino acid residues have been expanded:
Figure US11897926-20240213-C00008
The compound according to SEQ ID NO:11 is prepared substantially as described by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is used for the lysine at position 26 rather than at position 17 and the protected diacid is mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China). The molecular weight is determined by LC/MS (Found: [M+3H]3+=1653.4; Calc. [M+3H]3+=1653.52; Found MW (avg)=4957.2; Calc. MW (avg)=4957.57).
The compounds according to Example 8 (SEQ ID NO:12) through Example 122 (SEQ ID NO:126) are prepared substantially as described by the procedures of Example 1.
Found
SEQ Calculated (avg)
Example ID NO Compound Name (avg) MW MW
8 12 Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4926.61 4926.6
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-CO2H)AQ-(Aib)-
DFVNWLLAQGPSSGAPPPS-NH2
9 13 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4978.64 4978.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu-
CO-(CH2)18-CO2H)HQ-(Aib)-
DFVNWLLAQGPSSGAPPPS-NH2
10 14 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4950.59 4950.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)HQQDFVNWLLAGGPSSGAPPPS-
NH2
11 15 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4955.61 4955.2
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)AQQDFVNWLLAQGPSSGAPPPS-
NH2
12 16 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4884.53 4884.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)AQQDFVNWLLAGGPSSGAPPPS-
NH2
13 17 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 5035.7 5035.2
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)HQQEFVNWLLAQGPSSGAPPPS-
NH2
14 18 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 5036.68 5036.8
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)HQQDFVEWLLAQGPSSGAPPPS-
NH2
15 19 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4998.63 4998.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-
CO2H)HQQDFVNYLLAQGPSSGAPPPS-
NH2
16 20 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4899.54 4899.3
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
DFVEWLLAQGPSSGAPPPS-NH2
17 21 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4942.56 4941.9
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
DFVNWLLEQGPSSGAPPPS-NH2
18 22 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4846.48 4846.5
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-DFVN-(4-
Pal)-LLAQGPSSGAPPPS-NH2
19 23 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4813.45 4813.2
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
DFVNWLLAGGPSSGAPPPS-NH2
20 24 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4898.55 4898.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
EFVNWLLAQGPSSGAPPPS-NH2
21 25 Y-(Aib)-EGTFISDYSIALDKK((2-[2-(2- 4828.42 4827.9
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)HQ-(Aib)-DFVE-(4-
Pal)-LLAGGPSSGAPPPS-NH2
22 26 Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4870.5 4870.5
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)HQ-(Aib)-DFVE-(4-
Pal)-LLAGGPSSGAPPPS-NH2
23 27 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4842.45 4842.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)16-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
24 28 Y-(Aib)-EGTFISDYSI-(Aib)-LDKK((2-[2- 4842.45 4842.3
(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)16-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
25 29 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4907.56 4907.1
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVNAVLLAGGPSSGAPPPS-NH2
26 30 Y-(Aib)-EGT-αMeF(2F)- 4925.56 4925.4
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
EFVNWLLAGGPSSGAPPPS-NH2
27 31 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4841.5 4841.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
EFVQWLLAGGPSSGAPPPS-NH2
28 32 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4885.51 4885.2
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
EFVNWLLEGGPSSGAPPPS-NH2
29 33 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4842.49 4842.45
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
EFVEWLLAGGPSSGAPPPS-NH2
30 34 Acetyl-(D-Tyr)-AEGT-αMeF(2F)- 4902.47 4902.0
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)16-
CO2H)HQ-(Aib)-
EFVNYLLAGGPSSGAPPPS-NH2
31 35 Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4884.53 4884.6
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)HQ-(Aib)-
DFVNYLLAAGPSSGAPPPS-NH2
32 36 Y-(D-Ala)-EGTLISDYSILLDKK((2-[2-(2- 4850.51 4850.4
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)AQ-(Aib)-
DFVNWLLAQGPSSGAPPPS-NH2
33 37 Acetyl-(D-Tyr)-AEGT-αMeF(2F)- 4988.56 4988.1
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
EFVNYLLEGGPSSGAPPPS-NH2
34 38 Y-(Aib)-EGT-αMeF(2F)- 4946.57 4946.4
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
EFVNYLLATGPSSGAPPPS-NH2
35 39 Y-(Aib)-EGT-(αMeL)-ISDYSIALDKK((2- 4894.56 4894.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVNYLLATGPSSGAPPPS-NH2
36 40 Y-(Aib)-EGT-αMeF(2F)- 5005.59 5005.8
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
DFVEYLLETGPSSGAPPPS-NH2
37 41 Y-(Aib)-EGT-αMeF(2F)- 4961.54 4961.4
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
DFVEYLLEGGPSSGAPPPS-NH2
38 42 Y-(Aib)-EGT-αMeF(2F)- 4990.58 4990.2
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-DFVE-(4-Pal)-
LLETGPSSGAPPPS-NH2
39 43 Y-(Aib)-EGT-αMeF(2F)- 4946.53 4946.4
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-DFVE-(4-Pal)-
LLEGGPSSGAPPPS-NH2
40 44 Y-(Aib)-EGT-αMeF(2F)- 5028.63 5028.6
ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)HQ-(Aib)-
DFVEWLLETGPSSGAPPPS-NH2
41 45 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4972.59 4972.5
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLETGPSSGAPPPS-NH2
42 46 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4999.61 4999.8
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEQGPSSGAPPPS-NH2
43 47 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4942.56 4942.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEAGPSSGAPPPS-NH2
44 48 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4928.54 4928.1
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEGGPSSGAPPPS-NH2
45 49 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5014.67 5014.65
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLETGPSSGAPPPS-NH2
46 50 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5041.69 5041.35
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEQGPSSGAPPPS-NH2
47 51 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4970.62 4970.4
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEGGPSSGAPPPS-NH2
48 52 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4912.58 4912.8
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
49 53 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4884.51 4884.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)16-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
50 54 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4926.61 4926.9
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPPS-
NH2
51 55 Y-(Aib)-EGT-αMeF(2F)-ISDYSI-(αMeL)- 4944.6 4944.6
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPP5-
NH2
52 56 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-EFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
53 57 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4927.59 4927.5
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEYLLAGGPSSGAPPPS-NH2
54 58 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4950.63 4950.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEWLLAGGPSSGAPPPS-NH2
55 59 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4864.54 4864.8
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPPS-
NH2
56 60 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-D-
(αMeF)-VE-(4-Pal)-LLAGGPSSGAPPPS-
NH2
57 61 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4997.69 4997.5
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-EFVE-
(4-Pal)-LLAQGPSSGAPPPS-NH2
58 62 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4970.66 4970.4
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-EFVE-
(4-Pal)-LLATGPSSGAPPPS-NH2
59 63 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4940.63 4940.4
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-EFVE-
(4-Pal)-LLAAGPSSGAPPPS-NH2
60 64 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 5011.71 5011.5
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-EFVE-(4-Pal)-LLAQGPSSGAPPPS-
NH2
61 65 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4984.69 4984.8
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-EFVE-(4-Pal)-LLATGPSSGAPPP5-
NH2
62 66 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4954.66 4954.2
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-EFVE-(4-Pal)-LLAAGPSSGAPPPS-
NH2
63 67 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5070.73 5070.9
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVEYLLEQGPSSGAPPPS-NH2
64 68 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5043.71 5043.9
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVEYLLETGPSSGAPPPS-NH2
65 69 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5013.68 5013.9
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVEYLLEAGPSSGAPPPS-NH2
66 70 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5029.68 5029.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEYLLETGPSSGAPPPS-NH2
67 71 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5056.71 5056.8
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEYLLEQGPSSGAPPPS-NH2
68 72 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4999.65 4999.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEYLLEAGPSSGAPPPS-NH2
69 73 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4985.63 4985.7
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEYLLEGGPSSGAPPPS-NH2
70 74 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4956.63 4956.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLATGPSSGAPPPS-NH2
71 75 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5052.72 5052.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
DFVEWLLETGPSSGAPPPS-NH2
72 76 Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 5009.69 5009.7
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVEYLLETGPSSGAPPPS-NH2
73 77 Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 4965.64 4965.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-
EFVEYLLEGGPSSGAPPPS-NH2
74 78 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4922.57 4922.4
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS-
NH2
75 79 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4966.63 4966.8
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLETGPSSGAPPPS-
NH2
76 80 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)-LD- 4908.55 4908.6
(Orn)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS-
NH2
77 81 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)-LD- 4952.6 4952.4
(Orn)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLETGPSSGAPPPS-
NH2
78 82 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAAGPSSGAPPPS-NH2
79 83 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4983.66 4983.3
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAQGPSSGAPPPS-NH2
80 84 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5000.64 5000.4
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLETGPSSGAPPPS-
NH2
81 85 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5027.67 5027.7
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEQGPSSGAPPPS-
NH2
82 86 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4956.59 4956.45
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS-
NH2
83 87 Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4957.57 4957.8
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLATGPSSGAPPPS-NH2
84 88 Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4984.6 4984.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAQGPSSGAPPPS-NH2
85 89 Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4927.55 4927.2
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAAGPSSGAPPPS-NH2
86 90 Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4913.52 4913.1
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLAGGPSSGAPPPS-NH2
87 91 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4942.56 4942.8
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)16-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEAGPSSGAPPPS-
NH2
88 92 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4936.6 4936.2
LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)HQ-
(Aib)-DFVE-(4-Pal)-LLEAGPSSGAPPPS-
NH2
89 93 Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 4950.63 4950.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(4-Pal)-LLEAGPSSGAPPPS-NH2
90 94 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5013.64 5013.6
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(αMeY)-LLEAGPSSGAPPPS-NH2
91 95 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4999.61 4999.5
[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-CO2H)HQ-(Aib)-DFVE-
(αMeY)-LLEGGPSSGAPPPS-NH2
92 96 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 5056.71 5057.1
(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEQGPSSGAPPPS-NH2
93 97 Y-(D-Ala)-EGTFISDYSILLDKIAQ-(Aib)- 4884.53 4884.3
DF VNWK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)16-
CO2H)LAQGPSSGAPPPS-NH2
94 98 Y-(Aib)-EGT-αMeF(2F)- 4874.46 4874.4
ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2-
(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)16-
CO2H)LAGGPSSGAPPPS-NH2
95 99 Y-(Aib)-EGT-αMeF(2F)- 4960.55 4960.8
ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2-
(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-
CO2H)LEGGPSSGAPPPS-NH2
96 100 Y-(Aib)-EGTFISDYSIALDKIHQ-(Aib)- 4870.5 4870.2
EFVNYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LAGGPSSGAPPPS-NH2
97 101 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKIHQ- 4884.53 4884.6
(Aib)-EFVNYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LAGGPSSGAPPPS-NH2
98 102 Y-(Aib)-EGT-αMeF(2F)- 4902.52 4902.15
ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2-
(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γ-
Glu)-CO-(CH2)18-
CO2H)LAGGPSSGAPPPS-NH2
99 103 Y-(Aib)-EGT-αMeF(2F)- 4961.54 4961.4
ISDYSIALDKIHQ-(Aib)-DFVEYK((2-[2-
(2-Amino-ethoxy)-ethoxy]-acetyl)2(γ-
Glu)-CO-(CH2)18-
CO2H)LEGGPSSGAPPPS-NH2
100 104 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4984.64 4984.2
(Aib)-EFVE-(4-Pal)-K((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEGGPSSGAPPPS-NH2
101 105 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4971.6 4971.75
IHQ-(Aib)-DFV-(D-Glu)-YK((2-[2-(2-
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-CO2H)LEGGPSSGAPPPS-
NH2
102 106 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4985.63 4985.1
IHQ-(Aib )-EFV-(D-Glu)-YK((2-[2-(2-
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-CO2H)LEGGPSSGAPPPS-
NH2
103 107 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4943.55 4943.4
IHQ-(Aib)-DFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)16-CO2H)LEGGPSSGAPPPS-NH2
104 108 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4999.65 4999.2
IHQ-(Aib)-EFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEAGPSSGAPPPS-NH2
105 109 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5029.68 5029.2
IHQ-(Aib)-EFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LETGPSSGAPPPS-NH2
106 110 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5056.71 5056.8
IHQ-(Aib)-EFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEQGPSSGAPPPS-NH2
107 111 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4985.63 4985.4
IHQ-(Aib)-EFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEGGPSSGAPPPS-NH2
108 112 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4956.59 4956.3
IHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-CO2H)LEGGPSSGAPPPS-
NH2
109 113 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4970.62 4970.4
(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEGGPSSGAPPPS-NH2
110 114 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4942.56 4942.2
(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)16-CO2H)LEGGPSSGAPPPS-NH2
111 115 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4928.54 4928.4
IHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)16-CO2H)LEGGPSSGAPPPS-
NH2
112 116 Y-(Aib)-EGT-αMeF(2F)- 4946.53 4946.4
ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEGGPSSGAPPPS-NH2
113 117 Y-(Aib)-EGT-αMeF(2F)- 4960.55 4960.8
ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-COH)
LEAGPSSGAPPPS-NH2
114 118 Y-(Aib)-EGT-αMeF(2F)- 5017.6 5017.2
ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-CO2H)
LEQGPSSGAPPPS-NH2
115 119 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIRQ- 5004.63 5004.6
(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEGGPSSGAPPPS-NH2
116 120 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4990.61 4990.2
IRQ-(Aib)-DFVEYK((2-[2-(2-Amino-
ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-CO-
(CH2)18-CO2H)LEGGPSSGAPPPS-NH2
117 121 Y-(Aib )-EGTFISDYSI-(αMeL)-LDKIHQ- 4999.65 4999.8
(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEAGPSSGAPPPS-NH2
118 122 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIRQ- 5018.66 5018.4
(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-
ethoxy]-acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEAGPSSGAPPPS-NH2
119 123 Y-(Aib)-EGT-αMeF(2F)- 4988.57 4988.4
ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEGGPSSGAPPPS-NH2
120 124 Y-(Aib)-EGT-αMeF(2F)- 5002.59 5002.5
ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEAGPSSGAPPPS-NH2
121 125 Y-(Aib)-EGT-αMeF(2F)- 5059.64 5059.8
ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)-
K((2-[2-(2-Amino-ethoxy)-ethoxy]-
acetyl)2-(γ-Glu)-CO-(CH2)18-
CO2H)LEQGPSSGAPPPS-NH2
122 126 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4984.64 4984.80
IHQ-(Aib)-EFVE-(4-Pal)-K((2-[2-(2-
Amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)-
CO-(CH2)18-CO2H)LEAGPSSGAPPPS-
NH2
Binding Assays
Glucagon (referred to as Gcg or hGcg) is a Reference Standard prepared at Eli Lilly and Company. GLP-1(7-36)-NH2 (referred to as GLP-1 or hGLP-1) is obtained from CPC Scientific (Sunnyvale, CA, 97.2% purity, 100 μM aliquots in 100% DMSO). GIP(1-42)-NH2 (referred to as GIP) is prepared at Lilly Research Laboratories using peptide synthesis and HPLC chromatography as described above (>80% purity, 100 μM aliquots in 100% DMSO). [125I]-radiolabeled Gcg, GLP-1, or GIP is prepared using [125I]-lactoperoxidase and obtained from Perkin Elmer (Boston, MA).
Stably transfected cell lines are prepared by subcloning receptor cDNA into a pcDNA3 expression plasmid and transfected into human embryonic kidney (HEK) 293 (hGcgR and hGLP-1R) or Chinese Hamster Ovary (CHO) (hGIPR) cells followed by selection with Geneticin (hGLP-1R and hGIPR) or hygromycin B (hGcgR).
Two methods are used for the preparation of crude cell membranes.
Method 1: Frozen cell pellets are lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5, and Roche Complete™ Protease Inhibitors with EDTA. The cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes. The homogenate is centrifuged at 4° C. at 1100×g for 10 minutes. The supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer and rehomogenized as described above. The homogenate is centrifuged at 1100×g for 10 minutes. The second supernatant is combined with the first supernatant and centrifuged at 35000×g for 1 hour at 4° C. The resulting membrane pellet is resuspended in homogenization buffer containing protease inhibitors at approximately 1 to 3 mg/mL, quick frozen in liquid nitrogen and stored as aliquots in a −80° C. freezer until use.
Method 2: Frozen cell pellets are lysed on ice in hypotonic buffer containing 50 mM Tris HCl, pH 7.5, 1 mM MgCl2, Roche Complete™ EDTA-free Protease Inhibitors and 25 units/mL DNAse I (Invitrogen). The cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 20 to 25 strokes. The homogenate is centrifuged at 4° C. at 1800×g for 15 minutes. The supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer (without DNAse I) and rehomogenized as described above. The homogenate is centrifuged at 1800×g for 15 minutes. The second supernatant is combined with the first supernatant and centrifuged an additional time at 1800×g for 15 minutes. The overall supernatant is then centrifuged at 25000×g for 30 minutes at 4° C. The resulting membrane pellet is resuspended in homogenization buffer (without DNAse I) containing protease inhibitors at approximately 1 to 3 mg/mL and stored as aliquots in a −80° C. freezer until use.
Binding Determination Methods
The equilibrium binding dissociation constants (Kd) for the various receptor/radioligand interactions are determined from homologous competition binding analysis instead of saturation binding due to high propanol content in the [125I] stock material. The Kd values determined for the receptor preparations were as follows: hGcgR (3.9 nM), hGLP-1R (1.2 nM) and hGIPR (0.14 nM).
[125I]-Glucagon Binding
The human Gcg receptor binding assays are performed using a Scintillation Proximity Assay (SPA) format with wheat germ agglutinin (WGA) beads (Perkin Elmer). The binding buffer contains 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% (w/v) bacitracin (Research Products), 0.003% (w/v) Polyoxyethylenesorbitan monolaurate (TWEEN®-20), and Roche Complete™ Protease Inhibitors without EDTA. Peptides and Gcg are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves). Next, 5 μL serially diluted compound or DMSO is transferred into Corning® 3632 clear bottom assay plates containing 45 μL assay binding buffer or unlabeled Gcg control (non-specific binding or NSB, at 1 μM final). Then, 50 μL [125I]-Gcg (0.15 nM final), 50 μL human GcgR membranes (1.5 μg/well) and 50 μL of WGA SPA beads (80 to 150 μg/well) are added with a Biotek Multiflo dispenser. Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 12 hours of incubation/settling time at room temperature. Final assay concentration ranges for peptides tested in response curves is typically 1150 nM to 0.058 nM and for the control Gcg from 1000 nM to 0.05 nM.
[125I]-GLP-1 Binding
The human GLP-1 receptor binding assay is performed using an SPA format with WGA beads. The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, and Roche Complete™ Protease Inhibitors without EDTA. Peptides and GLP-1 are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves). Next, 5 serially diluted compound or DMSO is transferred into Corning® 3632 clear bottom assay plates containing 45 μL assay binding buffer or unlabeled GLP-1 control (non-specific binding or NSB, at 0.25 μM final). Then, 50 μL [125I]-GLP-1 (0.15 nM final), 50 μL human GLP-1R membranes (0.5 μg/well and 50 μL of WGA SPA beads (100 to 150 μg/well) are added with a Biotek Multiflo dispenser. Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 5 to 12 hours of incubation/settling time at room temperature. Final assay concentration ranges for peptides tested in response curves are typically 1150 nM to 0.058 nM and for the control GLP-1, 250 nM to 0.013 nM.
[125I]-GIP Binding
The human GIP receptor binding assay is performed using an SPA format with WGA beads. The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, and Roche Complete™ Protease Inhibitors without EDTA. Peptides and GIP are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves). Next, 5 μL serially diluted compound or DMSO is transferred into Corning® 3632 clear bottom assay plates containing 45 μL assay binding buffer or unlabeled GIP control (non-specific binding or NSB, at 0.25 μM final). Then, 50 μL [125I]-GIP (0.075-0.15 nM final), 50 μL human GIPR membranes (3 μg/well) and 50 μL of WGA SPA beads (100 to 150 μg/well) are added with a Biotek Multiflo dispenser. Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 2.5 to 12 hours of incubation/settling time at room temperature. Final assay concentration ranges for peptides tested in response curves is typically 1150 to 0.058 nM or 115 nM to 0.0058 nM and for the control GIP, 250 nM to 0.013 nM.
Binding Assay Data Analysis
Raw CPM data for concentration curves of peptides, Gcg, GLP-1, or GIP are converted to percent inhibition by subtracting nonspecific binding (binding in the presence of excess unlabeled Gcg, GLP-1, or GIP, respectively) from the individual CPM values and dividing by the total binding signal, also corrected by subtracting nonspecific binding. Data are analyzed using four-parameter (curve maximum, curve minimum, IC50, Hill slope) nonlinear regression routines (Genedata Screener, version 12.0.4, Genedata AG, Basal, Switzerland). The affinity constant (Ki) is calculated from the absolute IC50 value based upon the equation Ki=IC50/(1+D/Kd) where D is the concentration of radioligand used in the experiment, IC50 is the concentration causing 50% inhibition of binding and Kd is the equilibrium binding dissociation constant of the radioligand (described above). Values for Ki are reported as the geometric mean, with error expressed as the standard error of the mean (SEM) and n is equal to the number of independent replicates (determined in assays performed on different days). Geometric Means are calculated as follows:
Geometric Mean=10(Arithmetic Mean of Log Ki Values))
    • n=y/x means that only a subset of replicates (y) out of the total number of replicates (x) is used to express the mean. SEM is only calculated when y=2 or greater. Means are expressed as geometric means with the standard error of the mean (SEM) and the number of replicates (n) indicated in parentheses.
TABLE 1
In vitro Binding Affinity (Ki) of indicated Examples and comparator
molecules for human GcgR, GIPR, and GLP-1R in the presence of
0.1% bacitracin.
hGcgR hGIPR hGLP1R
Example or Ki (nM) Ki (nM) Ki (nM)
Comparator (SEM, n) (SEM, n) (SEM, n)
hGIP-NH2 1150 0.125 1100
(18.3, n = 4) (0.00511, n = 319) (143, n = 4)
hGlucagon 3.05 >2420 >4940
(0.120, n = 457) (n = 3) (n = 5)
hGLP-1 (7- >4590 >2300 0.785
36) NH2 (n = 5) (n = 3) (0.0252, n = 489)
1 228 0.0667 57.8
(46.6, n = 5) (0.0566, n = 5) (16.7, n = 5)
2 379 0.0373 77.9
(218, n = 3) (0.000430, n = 3) (20.2, n = 3)
3 338 0.0525 381
(220, n = 8) (0.0215, n = 8) (226, n = 8)
4 431 0.0620 423
(253, n = 6) (0.0259, n = 6) (235, n = 6)
5 619 0.0771 >913
(140, n = 9) (0.0342, n = 9) (n = 9)
6 453 0.104 571
(125, n = 7) (0.0866, n = 7) (368, n = 7)
7 >961 0.193 >913
(n = 5) (0.157, n = 5) (n = 5)
8 101 0.027 38.1
9 >230 0.110 168
10 >230 0.159 60.7
11 163 0.0584 85.9
12 134 0.0876 46.8
13 >230 0.237 >213
14 >230 0.460 >213
15 >254 0.471 >236
16 549 0.0438 99.2
17 274 0.0571 106
18 463 0.0331 158
19 335 0.0469 15.2
20 216 0.0477 79.8
21 >1530 1.46 801
22 >1380 0.219 388
23 323 0.0341 251
24 >960 0.254 729
25 101 0.0417 15.8
26 74.1 0.045 16.5
27 401 0.0354 23.2
28 86.8 0.0732 6.46
29 133 0.0569 22.7
30 407 0.037 26.5
31 >959 0.0473 266
32 >959 0.0343 85.2
33 63.5 0.0369 31.2
34 143 0.034 201
35 >960 0.0351 177
36 201 0.0683 393
37 51.4 0.026 83.9
38 182 0.0588 776
(59.8, n = 2) (0.0112, n = 2) (134, n = 2)
39 76.0 0.0444 235
(19.3, n = 2) (0.00439, n = 2) (8.34, n = 2)
40 50.9 0.0594 302
41 758 0.143 >906
42 99.1 0.0392 563
43 285 0.0366 605
(121, n = 2)
44 50 0.059 209
45 660 0.0663 >909
(257, n = 3) (0.0241, n = 3) (n = 3)
46 435 0.0319 744
(89.6, n = 3) (0.00724, n = 3) (58.2, n = 3)
47 175 0.0452 320
(69.7, n = 3) (0.0127, n = 3) (77.8, n = 3)
48 267 0.0498 211
49 >960 0.0512 596
50 23.4 0.0501 34.9
51 26.7 0.0386 54.1
52 114 0.0372 128
53 152 0.0184 62.9
54 386 0.0326 49.9
55 >960 0.0331 262
56 193 0.0487 530
57 422 0.0154 280
58 418 0.0324 459
(1.17, n = 2) (0.00694, n = 2) (68.3, n = 2)
59 230 0.0148 109
60 26.2 0.0390 51.4
61 80.4 0.0665 135
62 31.3 0.0414 47.6
63 185 0.0248 327
64 196 0.022 477
65 279 0.0316 305
66 279 0.0866 326
68 290 0.0948 421
69 114 0.0406 144
70 857 0.0421 570
71 102 0.0422 347
72 837 0.0594 437
73 292 0.036 117
74 511 0.0287 395
75 >958 0.0626 >902
76 358 0.0293 263
77 >958 0.0841 >905
(n = 2) (0.0207, n = 2) (n = 2)
78 505 0.0236 394
79 888 0.0229 486
80 >958 0.0828 >906
(n = 2) (0.0284, n = 2) (n = 2)
81 699 0.0374 714
(35.4, n = 2) (0.0106, n = 2) (39.8, n = 2)
82 305 0.0617 388
(112, n = 2) (0.00946, n = 2) (140, n = 2)
83 >958 0.116 452
84 >958 0.0584 325
85 800 0.0529 220
86 462 0.0533 108
87 576 0.0538 849
88 788 0.0830 261
89 611 0.0720 294
(n = 3) (0.0169, n = 4) (72.9, n = 4)
90 112 0.129 102
91 54.4 0.122 37.4
92 >958 0.0331 >902
93 >959 0.0430 >909
94 114 0.0310 >909
95 85.0 0.0255 832
96 >960 0.232 >908
97 77.5 0.0249 570
98 38.4 0.0195 311
99 174 0.0341 >901
100 572 0.0674 >913
(182, n = 4) (0.0265, n = 3) (n = 3)
101 >961 0.139 >913
(n = 2) (0.0585, n = 2) (n = 2)
102 >961 0.164 >913
(n = 3) (0.113, n = 3) (n = 3)
103 >960 0.0405 >913
104 >960 0.134 >910
105 >960 0.174 >910
106 304 0.0918 >910
107 347 0.0722 >911
108 >960 0.134 >910
109 >960 0.117 >908
110 >960 0.339 >908
111 >960 0.0871 >908
112 257 0.0742 >908
113 821 0.108 >921
(3.12, n = 4) (0.0287, n = 5) (n = 5)
114 >971 0.121 >921
(n = 3) (0.0496, n = 4) (n = 4)
115 373 0.0997 >912
(n = 2) (0.0472, n = 2) (n = 2)
116 178 0.166 >912
(n = 2) (0.0753, n = 2) (n = 2)
117 685 0.0722 591
118 NA 0.0696 >920
119 68.8 0.129 723
(n = 2) (0.0552, n = 2) (119, n = 2)
120 575 0.171 >912
(n = 2) (0.231, n = 2) (n = 2)
121 94.9 0.103 >912
(n = 2) (0.0473, n = 2) (n = 2)
122 957 0.0311 >913
As demonstrated in Table 1, examples of the present invention are very potent binders of the human GIPR, and have lower affinity for the GLP-1R and GcgR.
cAMP Pharmacological Functional Assay in Presence of 0.1% Casein
A set of cAMP assays are conducted in HEK293 cells expressing the human GLP-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR), or glucagon receptor (GcgR). Each receptor over-expressing cell line (20 μl) is treated with the test peptide in DMEM (Gibco Cat #31053) supplemented with 0.1% Casein (Sigma Cat #C4765), 250 μM IBMX, 1× GlutaMAX™ (Gibco Cat #35050), and 20 mM HEPES (HyClone Cat #SH30237.01) in a 20 μl assay volume. After incubating for 60 minutes at room temperature, the resulting increase in intracellular cAMP is quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ). The Lysis buffer containing cAMP-d2 conjugate (20 μl) and the antibody anti-cAMP-Eu3+-Cryptate (20 μl) are then added to determine the cAMP level. After incubating 60 minutes at room temperature, HTRF signal is detected with an Envision 2104 plate reader (PerkinElmer). Fluorescent emission at 620 nm and at 665 nm is measured and the ratio between 620 nm and at 665 nm is calculated and then are converted to nM cAMP per well using a cAMP standard curve. Dose response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four parameter non-linear regression fit with a variable slope (Genedata Screener 13). EC50 is the concentration of compound causing half-maximal simulation in a dose response curve. A relative EC50 value is derived by non-linear regression analysis using the percent maximal response vs. the concentration of peptide added, fitted to a four-parameter logistic equation.
Using Homogeneous Time Resolved Fluorescence methods, assays are conducted to determine the intrinsic potency of Example and comparator molecules performed in the presence of casein (instead of serum albumin) as a nonspecific blocker, which does not interact with the fatty acid moieties of the analyzed molecules.
Intracellular cAMP levels are determined by extrapolation using a standard curve. Dose response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four-parameter non-linear regression fit with a variable slope (Genedata Screener 13). EC50 is the concentration of compound causing half-maximal simulation in a dose response curve. Each relative EC50 value for the geometric mean calculation is determined from a curve fitting.
Concentration response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four-parameter non-linear regression fit with a variable slope (Genedata Screener 13). EC50 is the concentration of compound causing half-maximal simulation in a dose response curve. The EC50 summary statistics are computed as follows: Geometric mean:
    • GM=10(arithmetic mean of log10 transformed EC50 values). The standard error of the mean is reported:
    • SEM=geometric mean×(standard deviation of log10 transformed EC50 values/square root of the # of runs)×loge of 10.
    • The log transform accounts for the EC50 values falling on a multiplicative, rather than an arithmetic scale.
Each time the assay is performed, the test peptides are run plus the native ligands GIP, GLP-1, and glucagon, buffer only as baseline (minimum) and the highest concentration of the respective GIP, GLP-1, and glucagon standard is used as maximum for calculations. For illustration, as shown by Example 1, the peptide is tested in 8 runs of the hGIPR cAMP assay. For avoidance of doubt, hGIP amide, hGLP-1 amide, and glucagon EC50 in Table 2 are illustrative of geometric mean values from a series of 18 assay values, and values will vary each day compared to the zero buffer. Accordingly, each Example will use the geometric mean of those values to normalize the Example assay runs.
TABLE 2
Functional activation of hGcgR, hGIPR, and hGLP-1R in the presence
of 0.1% Casein.
hGcgR cAMP hGIPR cAMP hGLP1R cAMP
Example or Rel EC50 nM Rel EC50 nM Rel EC50 nM
Comparator (SEM, n) (SEM, n) (SEM, n)
hGIP-NH2 >5000 0.122 >500
(n = 5) (0.00449, n = 494) (n = 3)
hGlucagon 0.0116 9.79
(0.000315, (1.83, n = 3)
n = 306)
hGLP-1 (7- >500 0.0549
36) NH2 (n = 4) (0.00149, n = 490)
1 575 0.0476 >5000
(369, n = 4) (0.0253, n = 8) (n = 5)
2 608 0.0123 >5000
(n = 11) (0.00751, n = 15) (n = 11)
3 1250 0.0178 >5000
(398, n = 6) (0.00680, n = 9) (n = 6)
4 1690 0.0182 >5000
(n = 5) (0.00783, n = 5) (n = 5)
5 >5000 0.0148 >5000
(n = 5) (0.00434, n = 5) (n = 5)
6 >5000 0.0176 >5000
(n = 4) (0.00714, n = 14) (n = 4)
7 >5000 0.0219 >5000
(n = 2) (0.00294, n = 2) (n = 2)
8 295 0.0268 >5000
(62.9, n = 2) (0.000153, n = 3) (n = 2)
9 >5000 0.165 >100
10 >5000 0.611 >100
11 445 0.0738 >100
12 >5000 0.0911 >100
13 >5000 0.129 >100
14 >5000 0.340 >100
15 >5000 0.282 >100
16 >5000 0.00937 >5000
(n = 2) (0.00210, n = 2) (n = 2)
17 >5000 0.0135 >5000
(n = 2) (0.00227, n = 2) (n = 2)
18 >5000 0.00656 >5000
(n = 2) (0.00228, n = 2) (n = 2)
19 >5000 0.00992 >5000
(n = 2) (0.00148, n = 3) (n = 2)
20 >5000 0.00933 >5000
(n = 2) (0.00112, n = 3) (n = 2)
21 >5000 0.291 >5000
(n = 3) (0.0766, n = 4) (n = 3)
22 1250 0.0345 >5000
(487, n = 3) (0.00839, n = 4) (n = 3)
23 >5000 0.0192 206
(n = 5) (0.00529, n = 4) (13.8, n = 4)
24 >5000 0.0699 >5000
(n = 2) (0.0155, n = 3) (n = 2)
25 >5000 0.0155 8.39
(n = 2) (0.00237, n = 3) (2.46, n = 4)
26 >5000 0.00991 5.69
(n = 2) (0.00277, n = 2) (2.48, n = 4)
27 >5000 0.0132 >5000
(n = 2) (0.000266, n = 2) (n = 2)
28 >5000 0.0118 >5000
(n = 2) (0.00124, n = 2) (n = 2)
29 >5000 0.00679 >5000
(n = 4) (0.00193, n = 4) (n = 4)
30 >5000 0.00572 >5000
(n = 4) (0.00160, n = 4) (n = 4)
31 1750 0.0148 >5000
(886, n = 3) (0.00426, n = 3) (n = 3)
32 >5000 0.00469 >5000
(n = 2) (0.00199, n = 3) (n = 2)
33 >5000 0.0551 >5000
34 913 0.0177 462
(586, n = 2) (0.00478, n = 2) (321, n = 2)
35 >5000 0.0125 >5000
36 >5000 0.0543 1080
37 >5000 0.0360 61.1
38 >5000 0.0368 1390
39 >5000 0.0112 3330
40 >5000 0.0330 2330
41 >5000 0.0281 1800
42 >5000 0.0091 815
43 >5000 0.0154 1020
44 >5000 0.0084 1180
45 1690 0.0490 >5000
(n = 2) (0.000420, n = 2) (n = 2)
46 863 0.0294 >5000
(n = 2) (0.0166, n = 2) (n = 2)
47 757 0.0234 >5000
(492, n = 2) (0.00230, n = 2) (n = 2)
48 1360 0.0166 >5000
49 2860 0.0156 >5000
(620, n = 6) (0.00569, n = 7) (n = 6)
50 371.0 0.0212 41.0
51 308.0 0.0166 24.7
52 337.0 0.0194 >5000
53 344 0.0194 >5000
(136, n = 2) (0.00603, n = 3) (n = 2)
54 >5000 0.0540 >5000
55 >5000 0.0170 >5000
(n = 2)
56 >5000 0.0169 >5000
(n = 2)
57 886 0.0177 >5000
(430, n = 2) (0.00819, n = 3) (n = 2)
58 >5000 0.0183 >5000
(0.00544, n = 2)
59 >5000 0.0202 >5000
60 >5000 0.0369 71
61 >5000 0.0167 192
62 >5000 0.0116 58.2
63 1170 0.0398 >5000
64 3070 0.0448 >5000
65 850 0.0346 >5000
66 >5000 0.0786 >5000
67 >5000 0.0627 >5000
68 3030 0.0768 >5000
(n = 2) (n = 2)
69 803 0.0302 >5000
(237, n = 2) (0.00976, n = 2)
70 3560 0.0254 >5000
71 581 0.0721 >5000
72 >5000 0.0182 >5000
73 >5000 0.0151 >5000
74 627 0.0167 >5000
75 2170 0.0182 >5000
76 1200 0.0154 >5000
77 2660 0.0265 >5000
78 3000 0.0125 >5000
(n = 2) (0.00185, n = 2) (n = 2)
79 >5000 0.0316 >5000
80 >5000 0.0777 >5000
(n = 2) (0.0223, n = 2) (n = 2)
81 >5000 0.0282 >5000
(n = 2) (0.00791, n = 2) (n = 2)
82 3790 0.0391 >5000
(n = 2) (0.00658, n = 2) (n = 2)
83 >5000 0.0432 >5000
84 >5000 0.0340 >5000
85 >5000 0.0359 >5000
86 >5000 0.0300 >5000
87 >5000 0.0107 >5000
88 1670 0.0031 >5000
89 >5000 0.00687 >5000
(n = 2) (0.00245, n = 2) (n = 2)
90 >5000 0.0272 >5000
91 289 0.0321 530
92 >5000 0.0191 >5000
(n = 2) (0.00110, n = 2) (n = 2)
93 >5000 0.00482 884
(n = 2) (0.000315, n = 2) (n = 2)
94 >5000 0.00436 1090
(n = 4) (0.00186, n = 4) (442, n = 4)
95 >5000 0.0272 >5000
(0.0110, n = 2)
96 >5000 0.0251 >5000
97 >5000 0.0090 2510
98 >5000 0.00718 649
(n = 2) (0.00331, n = 3) (369, n = 2)
99 >5000 0.00454 >5000
(0.000120, n = 2)
100 >5000 0.0224 >5000
101 >5000 0.0396 >5000
(n = 2) (0.00639, n = 2) (n = 2)
102 >5000 0.0166 >5000
(n = 2) (0.00337, n = 2) (n = 2)
103 >5000 0.0129 >5000
(n = 2) (0.00901, n = 2) (n = 2)
104 >5000 0.0165 >5000
105 >5000 0.0179 >5000
106 >5000 0.0140 >5000
107 >5000 0.0199 >5000
108 >5000 0.0088 >5000
109 >5000 0.0113 >5000
110 >5000 0.0071 >5000
111 >5000 0.0065 >5000
112 >5000 0.0041 >5000
113 >5000 00.0142 >5000
(n = 3) (0.0108, n = 3) (n = 3)
114 >5000 0.0075 >5000
115 >5000 0.0372 >5000
116 >5000 0.0280 >5000
117 >5000 0.0617 >5000
118 >5000 0.0611 >5000
119 >5000 0.0220 >5000
120 >5000 0.0228 >5000
121 >5000 0.0196 >5000
122 >5000 0.0100 >5000
As demonstrated by data in Table 2, Example compounds of the present invention are very potent stimulating cAMP from human GIPR in the presence of 0.1% casein.
In Vivo Studies
Pharmacokinetics in male Cynomolgus Monkeys
The pharmacokinetics of select Examples are evaluated following a single subcutaneous administration of 50 nmol/kg to male cynomolgus monkeys. Blood samples are collected over 504 hours and resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K3 EDTA) concentrations are determined using a qualified LC/MS method that measured the intact mass of the compound. Each peptide and an analog as an internal standard are extracted from 100% cynomolgus monkey plasma using a protein precipitation method. Instruments are combined for LC/MS detection. Mean pharmacokinetic parameters are shown in Table 3.
TABLE 3
Mean Pharmacokinetic Parameters of peptides Following a
Single Subcutaneous Administration of 50 nmol/kg to
Male Cynomolgus Monkeys.
Cmax/Dose AUCInf/Dose
T1/2 Tmax (kg*nmol/ (hr*kg*nmol/ CL/F
Example (hr) (hr) L/nmol) L/nmol) (mL/hr/kg)
1 71.1 24 7.20 786 1.27
2 51.8 6 6.92 624 1.61
3 88.8 60 12.1 1764 0.57
4 124 6 8.71 1387 0.72
5 128 120 12.0 2262 0.44
6 129 6 7.83 1382 0.77
7 109 9 10.1 1603 0.63
Abbreviations:
T1/2 = half-life,
Tmax = time to maximal concentration,
Cmax/dose = maximal plasma concentration divided by dose,
AUCInf/Dose = AUCInf divided by dose,
CL/F = clearance/bioavailability.
Notes:
Data are the mean, where n = 2/group.
As seen in Table 3, results from this study for Example peptides tested are consistent with an extended pharmacokinetic profile.
Pharmacokinetics in Male Sprague Dawley Rats following Subcutaneous Administration
The pharmacokinetics of select Examples are evaluated following a single subcutaneous (SC) administration of 100 nmol/kg to male Sprague Dawley rats. Blood samples are collected over 168 hours following SC administration. Pharmacokinetic parameters are calculated using individual plasma concentrations. A qualified LC/MS method that measures the intact mass of the Example is used to determine plasma (K3 EDTA) concentrations. Each peptide and an analog as an internal standard are extracted from 100% rat plasma using a protein precipitation method. Instruments are combined for LC/MS detection. Mean pharmacokinetic parameters for the Examples are shown in Table 4.
TABLE 4
Mean (+/− SD) Pharmacokinetic Parameters of peptides Following a
Single Subcutaneous Administration of 100 nmol/kg to Male
Sprague Dawley rats.
Cmax/Dose AUCInf/Dose
T1/2 Tmax (kg*nmol/ (hr*kg*nmol/ CL/F
Example (hr) (hr) L/nmol) L/nmol) (mL/hr/kg)
1 37.4 24 3.62 280 3.57
2 26.9 12 5.47 304 3.30
3 24.9 12 3.03 130 7.67
5 27.1 24 4.61 239 4.20
6 34.8 24 4.07 271 3.69
Abbreviations:
T1/2 = half-life,
Tmax = time to maximal concentration,
Cmax/Dose = maximal plasma concentration divided by dose,
AUCInf/Dose = AUCInf divided by dose,
CL/F = clearance/bioavailability.
Notes:
Data are the mean, where n = 3/group, except for example 3, where the data is frpm n = 1 animal.
As seen in table 4, results from this study using these Example peptides are consistent with an extended pharmacokinetic profile.
In Vivo Effect on Insulin Secretion in Male Wistar Rats
Male Wistar rats with femoral artery and femoral vein canulas (Envigo, Indianapolis, IN) (280-320 grams) are single-housed in polycarbonate cages with filter tops. Rats maintained on a 12:12 h light-dark cycle (lights on at 6:00 A.M.) at 21° C. and receive food and deionized water ad libitum. Rats are randomized by body weight and dosed 1.5 mL/kg subcutaneously (s.c.) at doses of 0.3, 1.0, 3, 10, 30, and 100 nmol/kg 16 hours prior to glucose administration then fasted. Animals are weighed and anesthetized with sodium pentobarbital dosed intraperitoneally (i.p.) (65 mg/kg, 30 mg/mL). At time 0, a blood sample is collected into EDTA tubes after which glucose is administered intravenously (i.v.) (0.5 mg/kg, 5 mL/kg). Blood samples are collected for glucose and insulin levels at 2, 4, 6, 10, 20 and 30 min post-intravenous administration of glucose. Plasma glucose levels are determined using a clinical chemistry analyzer. Plasma insulin is determined using an electrochemiluminescence assay (Meso Scale, Gaithersburg, MD). Glucose and insulin AUC are examined compared to the vehicle control with n=5 animals per group. Results are presented (SEM)(N).
TABLE 5
The effect of Example compounds on insulin secretion
during intravenous glucose tolerance test.
Dose (nmol/kg, s.c.)
Ex-
ample 0.0 0.3 1.0 3.0 10 30 100
3 38.3 43.3 45.1 53.3  64.9 116.3 133.6
(8.8)(5) (0.8)(5) (8.8)(5) (6.5)(5) (9.3)(5) (28.6)(4) (16.1)(5)
5 35.9 36.2 45.3 80.6 122.0 144   212.7
(7.5)(5) (4.4)(5) (6.1)(5) (6.4)(5) (5.9)(5) (16.4)(5) (19.4)(5)
6 39.5 34.7 54.2 75.8 100.2 135.4 166.5
(2.2)(5) (3.6)(5) (6.8)(5) (10.3)(5) (15.6)(5) (22.9)(5) (21.6)(5)
The data provided by Table 5 demonstrate a dose dependent increase in insulin secretion.
TABLE 6
ivGTT Insulin Secretion shown by the following data:
Insulin secretion (ivGTT)
Example (ED50, nmol/kg) (SEM, n)
3 17.1 (n = 1)
5 18.4 (n = 1)
6 12.9 (n = 1)
    • The data provided by Table 6 demonstrate dose dependent increase in insulin secretion.
Immunogenicity Assessment of the Compounds of Examples 1, 2, 3, 5, and 6
The purpose of this study is to determine the relative potential for clinical immunogenicity of Example compounds 1, 2, 3, 5, and 6.
Methods:
CD4+ T Cell Assay: The CD4+ T cell assay is used to compare the compounds of Examples 1, 2, 3, 5, and 6 for a potential to induce an immune response in vivo according to methods known in the art (see, e.g., Jones et al. (2004) J. Interferon Cytokine Res. 24:560-572; and Jones et al. (2005) J. Thromb. Haemost. 3:991-1000), where an assessment of clinically tested monoclonal antibodies and peptides shows some degree of correlation between T cell proliferation observed in vitro and immunogenicity in the clinic. Protein therapeutics that induce less than 30% positive response in the CD4+ T cell proliferation assay are associated with a low risk of immunogenicity. Briefly, to assess the propensity fora clinical immunogenic response to the compounds of Examples 1, 2, 3, 5, and 6, CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) are prepared and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) from a cohort of 10 healthy donors with diverse human leukocyte antigen (HLA) class II haplotypes. Each donor is tested in triplicate with 2.0 mL media control, keyhole limpet hemocyanin (KLH; 0.33 μM), and the compounds of Examples 1, 2, 3, 5, and 6 (0.33 μM). Cultures are incubated for 7 days at 37° C. with 5% CO2. On day 7, samples are analyzed by flow cytometry using a BD LSR II Fortessa (Becton Dickinson; Franklin Lakes, NJ), equipped with a high throughput sampler (HTS). Data is analyzed using FlowJo® Software (FlowJo, LLC/TreeStar; Ashland, OR).
Results and Discussion
All donors produce a positive T cell response against KLH (100%). Analysis of the frequency and magnitude of the CD4+ T cell response for Example compounds is shown in Table 7.
TABLE 7
CD4 + T Cell Responses for Example compounds and
Positive Control (KLH).
Median Response
Example or % Donor Response Strength in positive
Comparator (n = 10) donors (CDI)
KLH 100%   164.285 (n = 10)
Example 1 0%   NA (n = 0)
Example 2 10%    2.69 (n = 1)
Example 3 0%   NA (n = 0)
Example 5 0%   NA (n = 0)
Example 6 0%   NA (n = 0)
    • Cell Division Index (“CDI”): proportion of divided CD4+ T cells to the total number of CD4+ T cells in stimulated versus unstimulated samples.
These data show that the frequency of positive CD4+ T cell response (CDI>2.5) was low for the tested Example compounds, and the magnitude of the response in the one positive donor from the Example 2 group was low (CDI<3). Thus, based on this assay, these compounds have a low risk of immunogenicity.
Amino Acid Sequences
GIP 1-42 (Human)
SEQ ID NO: 1
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ
GLP-1 (7-36) amide (Human)
SEQ ID NO: 2
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Glucagon (Human)
SEQ ID NO: 3
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
SEQ ID NO: 4
Z1X1X2EGTX6ISDYSIX13LDX16X17X18QX20X21X22VX24X25X26LX28X29
GPSSGAPPPSZ2

Claims (4)

We claim:
1. A compound, or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO:7.
2. A pharmaceutical composition comprising the compound, or pharmaceutically acceptable salt thereof, of claim 1 and at least one pharmaceutically acceptable carrier, diluent, or excipient.
3. A method for treating a condition selected from the group consisting of diabetes mellitus, obesity, and metabolic syndrome, in a patient in need thereof, comprising administering to the patient an effective amount of the compound, or pharmaceutically acceptable salt thereof, of claim 1.
4. A method for treating a condition selected from the group consisting of diabetes mellitus, obesity, and metabolic syndrome, in a patient in need thereof, comprising administering to the patient the pharmaceutical composition of claim 2.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013164483A1 (en) 2012-05-03 2013-11-07 Zealand Pharma A/S Gip-glp-1 dual agonist compounds and methods
WO2016066744A2 (en) 2014-10-29 2016-05-06 Zealand Pharma A/S Gip agonist compounds and methods
WO2016111971A1 (en) 2015-01-09 2016-07-14 Eli Lilly And Company Gip and glp-1 co-agonist compounds
US20180155407A1 (en) 2016-12-02 2018-06-07 Sanofi Compounds as peptidic glp1/glucagon/gip receptor agonists
WO2018181864A1 (en) 2017-03-31 2018-10-04 Takeda Pharmaceutical Company Limited Gip receptor activating peptide
WO2019125929A1 (en) 2017-12-21 2019-06-27 Eli Lilly And Company Incretin analogs and uses thereof
WO2020067575A1 (en) 2018-09-24 2020-04-02 Takeda Pharmaceutical Company Limited Gip receptor agonist peptide compounds and uses thereof
WO2020067557A2 (en) 2018-09-24 2020-04-02 Takeda Pharmaceutical Company Limited Gip receptor agonist peptide compounds and uses thereof
US11254721B2 (en) * 2019-08-01 2022-02-22 Eli Lilly And Company GIPR-agonist compounds

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013164483A1 (en) 2012-05-03 2013-11-07 Zealand Pharma A/S Gip-glp-1 dual agonist compounds and methods
WO2016066744A2 (en) 2014-10-29 2016-05-06 Zealand Pharma A/S Gip agonist compounds and methods
WO2016111971A1 (en) 2015-01-09 2016-07-14 Eli Lilly And Company Gip and glp-1 co-agonist compounds
US20180155407A1 (en) 2016-12-02 2018-06-07 Sanofi Compounds as peptidic glp1/glucagon/gip receptor agonists
WO2018181864A1 (en) 2017-03-31 2018-10-04 Takeda Pharmaceutical Company Limited Gip receptor activating peptide
WO2019125929A1 (en) 2017-12-21 2019-06-27 Eli Lilly And Company Incretin analogs and uses thereof
WO2020067575A1 (en) 2018-09-24 2020-04-02 Takeda Pharmaceutical Company Limited Gip receptor agonist peptide compounds and uses thereof
WO2020067557A2 (en) 2018-09-24 2020-04-02 Takeda Pharmaceutical Company Limited Gip receptor agonist peptide compounds and uses thereof
US11254721B2 (en) * 2019-08-01 2022-02-22 Eli Lilly And Company GIPR-agonist compounds

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Gault et al. (‘OPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP’ Cell Biology International v27 2003 pp. 41-46). (Year: 2003).
International Search Report of the International Searching Authority pertaining to International Application No. PCT/US2020/043988; dated Oct. 19, 2020; 8 pages.
Lorenz, M., Evers, A., & Wagner, M. (2013). Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity. Bioorganic & medicinal chemistry letters, 23(14), 4011-4018.
Written Opinion of the International Searching Authority pertaining to International Application No. PCT/US2020/043988; dated Oct. 19, 2020; 16 pages.

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